BIOLOGY 204/205 Advanced Genetics Laboratory

TABLE OF CONTENTS

Introduction………………………………………………………………..…..………….…p.2MODULE 1: Recombinant DNA…………………………………………...…….…..p.11MODULE 3: Proteomics………………………………………………………………….p.27Appendix A: Solutions Guide……………..………………………………....……...p.38

General guide…………………….……………………………p.38MODULE 1………………………….…………………………..p.40MODULE 2…………………………………….………………..p.44

Appendix B: Sterile Techniques.…………………………………………..…........p.48 Appendix C: Spread Plate Technique………………………………………….....p.48Appendix D: Pipette Use…………………………………………………………….....p.49Appendix E: Pipetting Exercise Pierce BCA Assay………………..……….…p.51Appendix F: GST Plasmid Map………………………………………………………..p.52Appendix H: Streak Plate Method………………………..………….…….......…p.53Appendix I: Frequently Used Ladders.…………………………….……………..p.54Appendix J: Southern Blot setup………………………….………..…..………....p.55Appendix K: PCR Chart for 1.17 ………………..…………………..……….….....p.56Appendix L: Chart for Making Sucrose …………………………..………………p.57Appendix M: Sucrose Gradient Tubes after Ultracentrifugation …...p.58Appendix N: Protein Gel Plate Setup ….……………………………………...…p.59Appendix O: Pierce BCA Protein Assay for Module 2.………………..…..p.60Appendix P: Assembling Protein Gel for Running Gels…..……….........p.61Appendix Q: Graphing Protein Data in Excel…………………………..……..p.62Appendix R: RPM to G-force Conversions……………………..………..……..p.63

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BIOLOGY 205Advanced Genetics Laboratory Spring Semester 2017

--- Introduction ---

Course Goals:

Master techniques of molecular biology Trouble shoot based on the student's knowledge of organic chemistry and biology Practice hypothesis development Improve written and oral expression Learn collaboration with lab members Keep a good laboratory notebook Cultivate an ability to develop protocols and experiments to test a hypothesis Become facile in graphing and spreadsheets in the analysis of data Become facile in statistical analysis of spreadsheet data Gain practical knowledge that will help with careers and jobs in research Put into practice the theoretical information that the student has learned through the

100 levels Become inspired to go on in biological science as a career

Biology 205 Advanced Genetics Laboratory --- Introduction ---

Biology 205 is a four credit course. We meet twice per week for 4 hours each. Our emphasis is on experimental design, techniques, data gathering and analysis. Work at the bench is given priority over work in a lecture setting. The modules are designed to approach real situations in ongoing research projects. Therefore, the modules are not necessarily designed to be finished in three hours. A few labs will run long, taking 5-6 hours to finish. A few labs will be relatively short. Students are expected to come into lab outside of the scheduled class time, usually at their own convenience, to perform short manipulations. Sometimes an experiment does not work and it has to be repeated. Coming to class well prepared and following directions carefully will cut down on potential mistakes!

We aim to help you learn to trouble shoot, think critically and analytically, use your knowledge of chemistry and physics and biology, and become comfortable with interpretation of your results.

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Module 1 Recombinant DNA/Bacterial Transformation

This module gives you some of the experience you would receive if you were to sub-clone a gene as a part of your research. That is, once you transform a bacterial line with the plasmid that you isolate, you will need to demonstrate that you have made the transfer of the correct gene.

Goals:1. Purify a plasmid and transform E. coli with the plasmid.2. Demonstrate that the transformants carry the plasmid by characterizing the

transformants’ phenotypes. 3. Analyze the size of the DNA plasmid in a cracking gel. 4. Hybridize with the original plasmid in a Southern blot.5. Amplify the gene inserted into the plasmid by PCR.6. Sequence part of the plasmid.

Module 2 Proteomics

In this module you will compare the proteins found in the membrane and axoneme of cilia.

You will run a one dimensional polyacrylamide gel, and cut the lanes of the gel from both the membrane and axoneme. The class will then compare the proteins identified in the two samples as well as compare the abundance of proteins between the samples.

Goals:1. Use ultracentrifugation to fractionate cells.2. Conduct a BCA Protein Assay to determine the concentration of proteins in samples

provided to you.3. Using SDS-PAGE, run a one dimensional gel and stain with Coomassie blue to visualize

differences in protein content of cilia types. 4. Cut out bands of interest from gel and prepare samples for mass spectrometry by

trypsinizing proteins.5. Analyze results from mass spec.

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Biology 205 Advanced Genetics Laboratory Grading Policy

Your grade will be based on the following components, each with approximately equal weight (16%):

1. Accuracy and quality of results from the lab experiments2. Lab citizenship* including working well and cooperatively with lab partner(s)3. Quality of participation in class discussions and student led reviews.4. Written report on a geneticist (50% of grade is on the draft; 50% on the final version)5. Presentation to the class about your report with your lab partner (last day of class or

during the scheduled exam time)6. Quality of your laboratory notebook

* Arriving on time and staying until work is complete. Doing work outside of class hours when necessary. Paying attention and being engaged during experiments. Following the safety rules, cleaning up, properly labeling your samples, putting materials away, etc.

*NOTE*: 10% will be deducted from the grade the day the assignment is late – an additional 10% is deducted for each additional 24 hours the assignment is late.

Each student will have a spread sheet kept by the instructor showing the grading for each week of laboratory work. The spread sheet will be sent to the student every two weeks. Note that some items are not graded every week, such as the notebook.

PlagiarismPlagiarism is taken very seriously and can be an expellable offense. If you plagiarize, you

will receive no credit for the assignment and could be brought to the attention of the University.

The University of Vermont has defined plagiarism as the following:

“All ideas, arguments, and phrases, submitted without attribution to other sources must be the creative product of the student. Thus, all text passages taken from the works of other authors (published or unpublished) must be properly cited. The same applies to paraphrased text, opinions, data, examples, illustrations, and all other creative work. Violations of this standard constitute plagiarism.” (University of Vermont, Vice Provost for Student Affairs, Policy V. 2.7.7)

If you are unsure of what constitutes plagiarism or if something is considered “paraphrased,” please come and see Dr. Van Houten for further explanation. Simply placing a citation and still copying the text or changing a few words within a paragraph that has been copied still constitutes plagiarism.

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For more resources and information on plagiarism, please see the following:University of Vermont policy:http://www.uvm.edu/policies/student/acadintegrity.pdfHarvard explanation with specific examples:http://isites.harvard.edu/icb/icb.do?keyword=k70847&pageid=icb.page342054

Laboratory Notebook Guidelines

We expect you to come prepared for class. A good method is to write the procedure for the day ahead of time in your laboratory notebook. Otherwise, you must write the procedure as you are carrying out the work. When changes occur, as in any research lab, they can be marked and initialed in your laboratory notebook. You are expected to have your lab notebook on your work bench, not your lab manual (the manuals can be referred when necessary).

Hard cover, bound notebook; no loose-leaf notebooks. Legibly record in blue or black ink – do not photocopy/print and paste the lab manual

into your notebook. Number all pages. Date all entries. Name, course number and email address should be on front cover. Reserve 3 pages at the beginning for the table of contents; keep up to date. Mistakes should be crossed out with a single line through the entry then initialed. Do not skip pages, do not rip pages out. Unused portions of a page should have a diagonal line drawn through the blank portion. Each experiment should begin on a new page. All data, calculations and graphs should be entered directly into the notebook.

Your notebook should provide enough detail so that another Advanced Genetics student could pick it up and repeat your procedure by following your entries. You should include all of the following information for each section:

Objective Who did the experiments (some procedures are done for you) Hazardous for that module All recipes for solutions used (Appendix A) Why were the procedures done Results Conclusions/discussion

A sample lab notebook is available in the lab for you to look at.

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Paper on a Geneticist

The purposes of writing this paper are to acquaint you with some of the world’s most innovative geneticists, give you practice with literature searching and citation, and provide you with an opportunity to communicate complex genetics concepts in a way that the lay public can understand.

You will work with your partner on researching a Nobel Laureate geneticist, whose name will be provided to you. You should go to the original literature; Wikipedia will not be accepted. The references you use should be shown to Dr. Van, who can also help to direct you to more if necessary.

Prepare a paper with the following structure:

▪ Introduction (1 page)▪ Short biography (1 page)▪ Description of the research for which the geneticist was awarded the Nobel

Prize (3 pages)▪ Discussion of why this research warranted a prize and how the research has

changed medicine or biomedical research in ways that impact the laypublic (2 pages)

Give the draft of the full paper to Dr. Van by March 2 at 2:40 pm for 50% of your grade; hand in the final on April 13 by 2:40 pm for 50% of your grade.

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General Lab Safety Rules:

1. Disinfect your bench top with a 10% bleach solution when you arrive and when you finish lab each day.

2. Wear gloves and avoid touching face and/or hair during an experiment. 3. Wash your hands before you leave lab.4. Do not eat, drink, smoke, chew gum or apply cosmetics while in lab.5. Dispose of all used materials as directed.6. Keep aisles clear – push in your chairs.7. Wipe all spills immediately. Inform lab tech and/or TA if you spill ANYTHING.8. Dispose broken glass in the appropriate receptacle. Inform lab tech and/or TA that you

have broken glass.9. Wear closed-toed shoes.10. Tie back long hair.11. Wear pants.12. Avoid wearing baggy, loose clothing or accessories that can interfere with your

experiment and/or may catch on fire. 13. Extinguish burners as soon as you finish using them.14. All Chemical Safety and MSDS information is located in the binder on the back of the

door.15. If you are unsure about a procedure, Please ask. 16. Cell phone, tablets, iPads, etc. are to be used only in the hallway. Please leave cell

phones outside the lab room. Points will be deducted for using cell phones and devices in the lab.

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Laboratory Exercise for First Day of Lab: Pipetting

Background:1. Acquaint yourself with the various denominations of pipettors in an attempt to avoid

mistakes, particularly when working under time pressure. (See Appendix C.)

2. The height of the fluid in the serological pipettes is measured at the bottom of the meniscus while the pipette is being held vertically.

3. Never put a pipette back into a sterile container.

4. Do not handle the lower part of the pipette.

Serial Dilutions: Serial dilutions allow you to dilute a sample many fold by making a series of small dilutions.

Standard Dilution Steps: Unless special circumstances demand it, the following are the only dilution steps that are used (For convenience and error avoidance in performing the accompanying arithmetic).

Concentration Dilution Concentrate/Dilutant Concentrate/Dilutant 10X 1:10 0.1 mL/0.9 mL 100 µL/900 µL

20X 1:20 0.1 mL/1.9 mL 50 µL/950 µL

50X 1:50 0.1 mL/4.9 mL 20 µL/980 µL

100X 1:100 0.1 mL/9.9 mL 10 µL/990µL

Pipette ExercisesPipette Exercise #1Determine and record the pipettor(s) best suited for each of the measurements listed below.

Volume Pipettor(s) Volume Pipettor(s) Volume Pipettor(s)10 µL 25 µL 0.0963 mL2 µL 0.015 mL 183 µL598.6 µL 0.200 mL 201.4 µL

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Pipette Exercise #2: Practice Pierce BCA protein AssayUse the chart provided (Appendix D) to develop a standard curve using BSA standards and to determine your unknown protein concentration. You will be setting up duplicates of Appendix E, running two sets of protein assays and averaging the OD values to calculate the unknown protein concentrations.

1. Obtain the unknown protein samples from your TA. 2. Add BSA and dH2O to the standard tubes according to the directions in Appendix D.3. Place 100 µL of the unknowns into the appropriate tubes. 4. Only after all your known and unknown tubes are prepared should you make the dye

solution: Use Solutions A and B from the Pierce BCA Protein Assay Kit. They should be mixed 50:1 (A:B). Show the calculations of the amount of dye you plan to make to the TA. (You should plan on extra tubes.) Mix the dye in the container provided.

5. Add 2 mL of the dye to each one of your samples and standards.6. Incubate at 37C for 30 minutes. 7. Get OD values for standards and samples: The TA will assist in the operation of the

spectrophotometer (spec). 8. Place 1 mL your first standard (blank) into a clean cuvette and use it to blank the spec.

Rinse the cuvette between each sample using dH2O. You may use more than one cuvette. Make sure to dry the outside of the cuvette with a Kimwipe™. Make sure the arrow on the top of the cuvette is facing toward you or away from

you. Handle the cuvette only on the top, smudges can interfere with the absorbance.

9. Read the OD at 562 nm. 10. Repeat to obtain the absorbance for each standard and unknown sample. Do the

readings for one complete set of standards and unknowns, then the second set of standards and unknowns. Do not blank the spec between each standard and unknown. Once the spec is blanked, do not blank again until you begin the second series.

11. Find the average OD’s of each standard and sample duplicates.12. Establish a standard curve using the OD values obtained with your BSA standards (See

Appendix E): graph Concentration (x-axis) vs. OD (y-axis) on the computers. Do not force the line through 0. Be sure to properly label your X- and Y- axis. Make sure there is a title on your graph. Include the slope equation and R2 value on the graph. Using the equation from the slope, calculate the unknown protein

concentrations of your unknowns. Send the final file to Ashik Nabi (Mnabi@uvm.edu) and yourselves. A copy of the

graph should be included in your lab notebook.

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Pipette Exercise #3 – We will let you know if you will carry out this exercise.Using the p1000 or the p200 pipettors, perform the following:

1. Set the pipettor to its maximum volume.2. Using water at room temperature, carefully pipette the water onto a weigh boat that

you have tared (re-zeroed).Room temperature water has a density of approximately 1 g/mL or 1 mg/L. Therefore, you can determine the accuracy of your pipetting, e.g., 1000 L of water will weigh 1 g. Repeat the pipetting until you feel that you are reasonably accurate. Then record the weights of five successive pipettings. Determine the mean and standard deviation associated with your measurements.

Complete the following conversions:1 L =_______mL 0.001 L =________mL10 L =_______mL 0.11 L =________mL100 L =_______mL 0.01 L =________mL1000 L =_______mL 1.0 L =________mL

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Module 1 Recombinant DNA

*Please refer to pages 2 & 3 for introduction and Appendix B for sterile techniques.

**Note: The E.coli cell line used in this module is K12. The K12 bacteria cells are to be transformed with the GST plasmid (Appendix F).

1.0 Overnight (ON) Bacterial Culture (Done for you) __________ 1. The lab tech will add 2.5 mL of bacteria cells previously grown ON to 100 mL LB-AMP

medium (per group).2. The cells will grow while shaking at 37°C ON.

1.1 Isolation of Plasmid DNA __________ HAZARDOUS CHEMICAL INFO: -Salt-Saturated Phenol (SS Phenol) is to be used only while wearing gloves under the hood. Dispose of all plastic and liquid waste containing SS Phenol in appropriate disposal containers in the fume hood. -Chloroform: Isoamyl Alcohol (24:1) (C:IA) is to be used only while wearing gloves under the hood. Dispose of all plastics and liquid waste containing C:IA in appropriate disposal containers in the fume hood.

1. Transfer the entire 100 mL overnight bacterial culture (transformed E. coli) to a large, sterile, screw top centrifuge bottle and harvest the bacteria by centrifuging at 5000 rpm, 4°C for 10 minutes (Beckman J2-21; JA-14 rotor). (For centrifuge conversions from rpm to × g or RCF, see Appendix O.)

2. Decant the supernatant broth into the waste jar.3. Resuspend the bacterial pellet in 5 mL of Solution I containing 5 mg/mL lysozyme.4. Transfer to a 30 mL polycarbonate screw top Oakridge centrifuge tube. Let stand at

room temperature for 5 minutes.5. Add 10 mL of freshly made Solution II. Place the cap on the tube and mix the contents

by inverting the tube several times, mixing gently. Let stand on ice for 10 minutes.6. Add 8 mL of ice-cold 3M potassium acetate, pH 4.8. Fill tubes only ¾ full. Screw on the

cap and mix by inverting. Let stand on ice for 30 minutes. (Total volume is 22 – 23 mL.)7. Balance the tubes before centrifugation.8. Centrifuge in the Beckman J2-21 (JA-17 rotor) at 15,000 rpm, 4°C for 20 minutes. The

genomic DNA and bacterial debris should form a tight pellet at the bottom of the tube. 9. Being very careful not to disturb the pellet, use a 10 mL pipet to transfer all of the

supernatant to a 30 mL glass screw top tube. Only take clear supernatant. 10. Add 1 mL of heat treated RNase A to each tube. Please use all of the RNase A provided. 11. Incubate at 37°C for 10 minutes in Innova 4000 then invert 2 to 3 times. Incubate and

additional 10 minutes in the Innova 4000.

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12. In the fume hood, add one volume of SS phenol using glass pipettes. (Note the yellow color which helps you identify the phenol phase in the next step.) Your tubes can be no more than 2/3 full (12 mL) including the addition of the phenol, so divide your original solution as necessary into 3 or 4 conical tubes, using glass pipettes. SAFETY NOTE: Phenol can cause severe burns to skin and damage clothing. Gloves and safety glasses should be worn when working with phenol. All manipulations should be carried out in the fume hood. A glass receptacle is available exclusively for disposing of used phenol and chloroform.

13. Vortex the conical tube and contents with lids on for 1 minute; be sure the contents are thoroughly mixed. Make sure the tops of the conical tubes are screwed on tightly to ensure that no leaking will occur. Centrifuge for 1 minute at 2800 rpm using the Eppendorf Centrifuge 5702.

14. Transfer the non-colored upper aqueous phase to a fresh conical tube. Do not take the interface, which is denatured protein. In the hood, add 1 volume of chloroform: isoamyl alcohol (24:1) – the same amount as the phenol you added. Vortex 1 minute and centrifuge 1 minute at 2800 rpm in the Eppendorf Centrifuge 5702.

15. Transfer the upper, aqueous layer to a fresh 30 mL glass tube and add 2.5 volume of cold 95% ethanol, using glass pipettes. You need to calculate how much total liquid will be in each 30 mL glass tube. The tube cannot be more than 2/3 full (20 mL), so you may have to use more than one 30 mL glass tube. Make your calculations before adding the ethanol!

16. Mix and allow it to precipitate in the -80°C freezer overnight.

1.1b Wash Pellets 17. Balance your tubes along with their rubber sleeves.18. Recover the DNA by centrifuging the tube at 4°C in the Beckman J2-21 (JA-17 rotor) at

9500 rpm for 30 minutes. 19. Discard the supernatant into a waste container. The pellet will look like a whitish residue

on the side of the tube. 20. Resuspend the pellet with 1 mL 70% ethanol by pipetting up and down onto the sides of

the tube. Try to resuspend the entire pellet to increase your plasmid yield. Transfer the resuspension from the first tube to the next tube until all pellets are resuspended and pooled together. Transfer the solution into one sterile 1.5 mL microfuge tube.

21. Centrifuge for 5 minutes at 14,000 rpm in the Eppendorf 5415C. Pipet out the ethanol; add 1 mL more of 70% ethanol to wash the pellet and vortex for 30 seconds. Spin at 14,000 rpm for 5 minutes using the Eppendorf 5415C.

22. Discard the ethanol; dry the pellet using the SpeedVac in the basement or leaving the tube in the fume hood overnight with the lid open. Give your sample to the TA/Lab tech to be properly dried for 15 – 20 minutes.

23. Store the dry pellet at -20°C.

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1. 3 Agarose gel to confirm isolation of the plasmid _____ HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.-UV light is very harmful if looked at directly. When viewing your gels on the UV light box be sure to wear a protective face mask, or place the shield on top of the box before turning on light.

1. Dissolve 0.35 g agarose in 50 mL 1X TAE buffer to make a 0.7% gel.2. Microwave on high for 1 minute. Be sure to place a Kimwipe™ in the opening of the

flask to help prevent evaporation.3. Swirl the flask and make sure all of the agarose is dissolved. If not, microwave more

until it is. Remove flask with a hot mitt. 4. Place the running tray into the gel-casting tray. Add comb. 5. Cool agarose slightly; approximately 5 minutes, swirling occasionally. Slowly pour

agarose into the farthest corner from the comb in the gel casting set up. Try to avoid bubbles! If bubbles persist, remove or pop them with a pipette tip. Let cool until opaque (30 minutes).

6. While your gel is setting, remove your DNA from the -20°C. Resuspend the pellet thoroughly in 300 µL of TE buffer. Remove 5 µL and place it in a 0.5 mL tube for the TA to check your DNA concentration. Divide the remaining final suspension into three labeled 1.5 mL microcentrifuge tubes.

7. Just before you are ready to load the gel, heat the λ Hind III marker (Appendix G) for 7 minutes in the 65°C hot block – Place on ice immediately.

8. Once your gel is set, remove it from the casting tray. Place it in the running tray, with the comb still set. Cover the gel with 1X TAE. Gently remove the comb. Removing the comb last will ensure that your wells do not collapse.

9. Prepare two aliquots of your plasmid: o For the first aliquot, mix 4 µL of 6X DNA loading buffer with 20 µL plasmid. o For the second aliquot, prepare a 1:5 dilution of your plasmid in TE buffer. The

volume should be 20 µL which should then be mixed with 4 µL of 6X DNA loading buffer.

10. You must also prepare 1X DNA loading buffer for all empty lanes. Each empty lane should get 20 µL 1X DNA loading buffer.

11. Be prepared to load the gel quickly—you do not want your DNA to diffuse into the running buffer.

12. Load all 24 µL of the plasmid DNA sample, 24 µL of the 1:5 diluted plasmid DNA sample and 20 µL of λ Hind III marker in the wells; put the lid on the box so that the DNA will run toward the red electrode.

13. Run the gel at 100V for ~1 hour.14. Stain the gel for approximately 15 minutes in ethidium bromide, followed by a brief

rinse in water.

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15. Examine the gel on the UV light box. If the ladder is not visible or is faint, place the gel back into the stain. When you feel that your gel is properly stained, the technician will take a picture to document your results.

16. Leave the gel in destain to be discarded later. 17. Rinse electrophoresis unit with RO water after use so the buffer does not dry on the

electrodes.

1.3 Grow an overnight broth culture of E. coli ( Done for you) __________ 1. A single colony of K12 E. coli is lifted from a nutrient agar plate and added, using a

sterile toothpick, to a sterile test tube containing 10 mL Luria Broth (LB).2. Culture is allowed to shake at 37°C overnight.

1.4 Transformation _____ Three hours before class the tech will remove 1 mL from the ON culture and use the 1 mL to inoculate 50 mL of fresh LB broth. It will shake at 37°C for two hours. This will produce cells in exponential growth phase for you to make competent and transform.

1. Divide broth culture into two sterile 30 mL screw top Oakridge centrifuge tubes; place tubes in ice for 30 minutes.

2. Thaw out one tube of your plasmid DNA on ice.3. Centrifuge the cultures at 4°C in the Beckman J2-21 (JA-14 rotor) for 10 minutes at 5000

rpm. Decant the supernatant into the collection flask provided.4. Resuspend one pellet in 25 mL ice cold 50 mM CaCl2. Combine this resuspension

solution with the second bacterial pellet; place on ice for 20 minutes. Keep CaCl2 on ice while waiting.

5. Centrifuge the cell suspension at 4°C in the Beckman J2-21 (JA-17 rotor) for 10 minutes at 5000 rpm.

6. Decant the supernatant and gently resuspend the pellet in 3 mL ice-cold 50 mM CaCl2 by pipetting up and down with the serological pipette.

7. Keep the cells on ice for 5 minutes.8. Place two aliquots of the chemically competent bacteria cells, 0.3 mL each, into ice-cold

1.5 mL microfuge tubes.9. Add 0.2 mL of transformation buffer to each tube. Save remaining competent cells at

4°C.10. Add 5 µL plasmid DNA to one tube. Add 5 µL TE buffer to the second tube. The second

tube will not receive plasmid DNA and will act as a control. Mix gently and leave on ice for 20 minutes.

11. Heat shock cells for 1 min in the 42°C water bath.12. Plunge tubes into ice and let sit on ice for 5 minutes. 13. Add 0.7 mL LB to each tube and tap gently with finger.14. Shake at 37°C for 60 minutes in Innova 4000.

**NOTE: during this hour incubation your TA or Lab Tech will demonstrate proper spreading and streaking procedures for plating (See Appendices B, H, and I). It is very

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important that you understand sterile techniques when working with bacteria so you don’t contaminate your samples.

15. Plate 0.05, 0.1, and 0.3 mL of the cells with plasmid DNA onto LB-AMP plates. Use the spread plate technique (Appendix H). Let the plates dry (covered) for 5 minutes right-side up before inverting the plates and placing in the incubator.

16. Streak (Appendix I) am small amount of the contents of the “no DNA tube” on an LB-AMP plate and on an LB plate. The LB-AMP plate will act as a negative control, while the LB plate will serve as a positive control.

17. Label plates appropriately with group color, date, type of bacteria, and any other important information.

18. Incubate the plates at 37°C overnight (upside down). Be sure to remove, wrap with Parafilm, and refrigerate the plates tomorrow!

Following Day: 1.5 Selecting for bacteria that carry the plasmid ______________ 1. Examine transformed and no DNA control plates. (There should be no colonies on the

“No DNA” LB-AMP plate, why?)2. Choose 3 well isolated colonies from the transformed plates. Streak (Appendix I) each

colony on an LB-AMP plate. 3. Choose 2 well isolated colonies from the control (non-transformed) plate provided.

Streak each colony on one LB plate.4. Incubate the plates overnight at 37°C.5. Wrap the old plates in Parafilm and refrigerate.

1.6 _ Secondary selection of transformed bacteria __________________________ 1. Transfer 2 well-isolated colonies from 2 different transformed streaks and place each in

its own 1 mL saline aliquot. Repeat with 1 control colony into its own 1 mL aliquot of sterile saline. Parafilm and refrigerate the old plates.

2. For the transformed bacteria, streak (Appendix I) 1 loop full of saline/bacteria suspension onto an LB-AMP plate. Do this for each of the 2 samples.

3. For the control cells, streak 1 loop full of the saline/bacteria suspension onto an LB plate. Be sure to label plates clearly!

4. Incubate at 37°C overnight. The following day, remove and Parafilm the plates before placing them at 4°C for the next day.

5. Go to 1.16

1.7 Preparing bacteria for the cracking gel (day before 1.8) _________________ 1. Using a marker, draw a line down the center of a new LB-AMP plate. Make a template

on paper with 1.5 cm x 1.5 cm squares on each half. Place the plate over the template.2. Choose 2 LB-AMP plates from Day 1.6 that show the best growth with isolated colonies.

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Using a loop, after sterilizing and briefly cooling it on the plate, select one colony from the Day 1.6 plate and “fill in” the square on the agar on the plate. Repeat for the 2nd colony using the loop, again sterilizing it and cooling it before selecting a colony.

3. Repeat the procedure for the control, but use a fresh LB plate.4. Incubate at 37°C overnight for at least 24 hr, but less than 36 hr. 5. Parafilm and refrigerate old plates.

1. 8 Next day: Cracking gel _____ HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Use a sterile loop or toothpick to scrape bacteria from the plates prepared the day before. Add bacteria from each square to 250 µL of cracking buffer (one square for one tube of 250 µL of cracking buffer). Do this for transformed and non-transformed cells (you should have a total of 2 microcentrifuge tubes). Vortex tubes to mix well.

2. Incubate the vortexed bacteria samples at 37°C in the hot water bath for 25 minutes.3. Prepare 0.7% agarose gel.4. Centrifuge the bacteria samples for 15 minutes at 14,000 rpm.5. Use a toothpick to remove the bacterial debris from the bottom of each tube. (You

won’t be able to see a pellet, but when you pull it out, it will look like a blue glob.)6. Load the gel slowly and carefully: 7. Lane 2: 24 µL plasmid DNA solution (Use the same DNA plasmid and DNA loading buffer

dilution you used in 1.2 that produced the best bands).Lane 3: 20 µL Hind III marker (Heat in 65C hot block for 7 minutes before loading) Lane 4: 20 µL 1X DNA loading bufferLane 5: Transformed supernatant Lane 6: 50 µL cracking buffer only Lane 7: Non-transformed supernatant Note: Load as much transformed and non-transformed supernatant as possible (A well-formed well can hold ~50 L).All empty lanes (1, 4, & 8) should receive 20 µL 1X DNA loading buffer.

8. Run the gel for ~1 hour at 100 volts.9. Stain with ethidium bromide, destain, and image.

1.9 Labeling DNA with Biotin __________ HAZARDOUS CHEMICAL INFO: -Salt-Saturated Phenol (SS Phenol) is to be used only while wearing gloves under the hood. Dispose of all plastic and liquid waste containing SS Phenol in appropriate disposal containers in the fume hood. -Chloroform is to be used only while wearing gloves under the hood. Dispose of all plastics and liquid waste containing chloroform in appropriate disposal containers in the fume hood.

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Part A: Labeling Reaction1. Remove an aliquot of your plasmid DNA from the -20°C and place on ice. 2. You should have the concentration of your plasmid and will need to calculate how much

of your plasmid is required to obtain 20 µg.3. Add the labeling reaction components to a 0.5 mL tube (on ice) in the following order:

dNTP mix 30 µL1X DNase I in Buffer 20 µLPlasmid DNA X µl (calculate volume for 20 µg)DNA Polymerase I 20 µLBring reaction up to 200 µL with dH2O

4. Mix well and centrifuge for 5 seconds in your desktop minifuge.5. Allocate 50 µL into four 0.5 mL tubes.6. Incubate at 15°C for 2 hours in thermocycler. 7. Add 5 µL Stop Buffer to each tube and mix.8. Incubate tubes at 65°C for 5 minutes in thermocycler.

Part B: Purification of DNA probe1. Transfer liquid to consolidate solution from the four 0.5 mL tubes into one 1.5 mL tube.2. Add 4 µL 10% SDS to the tube and mix.3. Add 110 µL Chloroform and 110 µL SS Phenol to an empty 1.5 mL microcentrifuge tube.4. Transfer DNA solution to the tube containing chloroform and phenol. Close tightly and

vortex for 2 minutes.5. Centrifuge for 2 minutes in the Eppendorf 5415C at 14,000 rpm.6. Collect the top layer of liquid and transfer to a fresh 1.5 mL microcentrifuge tube.

Discard the remaining liquid into waste container.7. Add 220 µL chloroform to tube. Vortex for 2 minutes and then centrifuge for 2 minutes

at 14,000 rpm in the Eppendorf 5415C.8. Collect top liquid layer and transfer to a clean tube. Discard the remaining liquid into

waste container. 9. Add 40 µL 3 M sodium acetate (pH 4.8) and 800 µL cold 95% ethanol. Mix gently by

inverting tube.10. Store at -20°C ON (at least 6 hours).

1.9b The Next Day:11. Centrifuge the sample for 5 min at 14,000 rpm in the Eppendorf 5415C (all centrifuging

here is done in this machine).12. Carefully remove the supernatant.13. Resuspend the pellet in 1 mL cold 70% ethanol. Centrifuge for 5 minutes at 14,000 rpm.

(1st Wash.)14. Remove supernatant (ethanol). 15. Resuspend the pellet in 1 mL cold 70% ethanol. Centrifuge for 5 minutes at 14,000 rpm.

(2nd wash.)16. Remove supernatant (ethanol). Let tube dry in the fume hood with the lid open

overnight.17. The following day, the sample will be moved to -20°C until use.

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1.10 Preparing for the Southern Blot (day before 1.11) __________ 1. Using a marker, draw a line down the center of a new LB-AMP plate. Make a template

on paper with 1.5 cm x 1.5 cm squares on each half. Place the plate over the template.2. Choose 2 LB-AMP plates from Day 1.6 that show the best growth with isolated colonies.

Using a loop, after sterilizing and briefly cooling it on the plate, select one colony from the Day 1.6 plate and “fill in” the square on the agar on the plate. Repeat for the 2nd colony using the loop, again sterilizing it and cooling it before selecting a colony.

3. Repeat the procedure for the control, but use a fresh LB plate.4. Incubate at 37°C overnight for at least 24 hr, but less than 36 hr. 5. Parafilm and refrigerate old plates.

1. 11 Southern Blot _____ HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Run cracking gel as you did before: Set up your gel just as you did for 1.8, except use 20 µL of plasmid mixed with 4 µL 6X DNA loading buffer for the plasmid lane regardless of previous results.

2. Stain the gel with ethidium bromide, briefly destain, and image the gel. 3. After imaging, make sure to destain the gel for 5 minutes before moving forward.4. Denature the gel in 0.5 M NaOH/0.8 M NaCl for 30 minutes while rocking at room

temperature. Decant the denaturing solution.5. Repeat step 4 , adding fresh denaturing solution and rock for 30 minutes.6. Decant the denaturing solution and rinse gel in dH2O for 1 minute.7. Neutralize the gel in 0.5 M Tris/1.5 M NaCl (pH 7.0) for 30 minutes while rocking. Decant

the neutralization solution.8. Repeat step 7 , adding fresh neutralization solution and rock for 30 minutes.9. While the gel is neutralizing, also prepare the Test Spot:

a. Take your Biotin labeled probe out of the freezer and resuspend it with 12 µL TE buffer.

b. Keep probe on ice. c. Cut a piece of nitrocellulose membrane approximately 2 cm x 2 cm. Make

sure to cut your test spot in a unique shape so that you can identify it later. For example, you can cut one or two small notches on the side of the square, cut off a corner of the nitrocellulose, or cut a unique shape.

d. Hydrate the nitrocellulose test spot in dH2O for 3 minutes.e. Soak the nitrocellulose test spot in 10X SSC for 3 minutes. f. Remove the nitrocellulose from the SSC and place on a small Kimwipe™ for 5

min.g. Add 2 µL of probe to the center of the square (or uniquely shaped) piece of

nitrocellulose.

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h. Allow the probe to dry for at least 10 min while the nitrocellulose test spot rests on the Kimwipe™, then wrap the nitrocellulose test spot in plastic wrap and store at -20°C until 1.12.

10. While the gel is in the second neutralization wash, cut and hydrate your nitrocellulose membrane for 3 minutes in dH2O, then in 10X SSC until your blot set-up is ready. Always wear gloves when handling the nitrocellulose. Always handle the membrane with forceps, and only around the edges so as to not create blotches of background color.

11. After the gel has finished the second 30 minute neutralizing step, rinse the gel in 10X SSC for 3 minutes while rocking.

12. Assembling the Southern Blot (Appendix J): a. First the wick (a long strip of Whatman 3M filter paper) needs to be placed on the

platform so that it can only touch the buffer on two sides.b. Place three pieces of Whatman 3M filter paper cut to the dimensions of your gel on

top of wick. c. The gel should be placed on top of the filter paper and the nitrocellulose should be

laid on top of that. **Make sure the nitrocellulose and the gel are lined up**d. Place three more pieces of Whatman 3M filter paper cut to the dimensions of your

gel on top of the nitrocellulose.e. Roll the filter paper with a test tube while applying gentle pressure to remove any

air bubbles. f. A stack of cut paper towels that are cut to the dimensions of your gel, that when

compressed is at least 10 cm high, should be assembled and placed on top of the filter paper.

g. Make sure the glass dish, or base, is filled with sufficient 10X SSC.h. Wrap the whole set up in plastic wrap to provide stability to the stack and minimize

evaporation. i. Pressure should be applied to the top of the stack to enhance wicking overnight. (Your TA should demonstrate this and assist in the assembly)

13. Let Southern blot transfer ON.

1.12 Drying of Blot (Done for you ) _____________________________________________ 1. Disassemble the Southern blot and rinse the nitrocellulose in 5X SSC for 2 minutes.2. Dry the nitrocellulose on a large Kimwipe™.3. Remove the nitrocellulose test spots from the -20°C freezer and remove the plastic

wrap.4. Bake nitrocellulose blot and the test spot on a Kimwipe™ in a vacuum oven at 80°C for 2

hours.5. Carefully place blot into hybridization bag and seal on all four sides. Carefully re-wrap

the baked nitrocellulose test spot in plastic wrap.6. Store blot and test spot in -20°C freezer.

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1.13 Hybridization of the Southern Blot _____ 1. Carefully unwrap your test spot. 2. Place your uniquely cut nitrocellulose test spot in a small plastic tub containing all of the

test spots from the class. Your TA or lab tech will hydrate these in 2X SSC and then place them in prehybridization solution and return them to you tomorrow for 1.14.

3. Cut a corner of your hybridization bag. Using a serological pipette, add 50 mL 2XSSC to the bag to hydrate your blot. Try to remove all the air bubbles and reseal the corner of the bag using the food sealer. The blot should be uniformly hydrated after several minutes.

4. While the blot hydrates, denature 200 L of Herring sperm DNA (2 mg/mL) in 100°C hot block for 10 minutes followed by plunging the tube into ice water.

5. For prehybridization of the nitrocellulose blot, add the 200 µL of freshly denatured Herring sperm to the prehybridization solution and mix.

6. Cut a corner of your bag and pour out the 2X SSC. Using a serological pipette, add the prehybridization solution (~8 mL). Reseal the corner of the bag after making sure to push all of the air bubbles out of the bag. If there are still more air bubbles in the bag after you have resealed the corner, set the bag upright and push all the bubbles to the top of the bag. Reseal the bag across the top to trap the air bubbles away from the blot.

7. Incubate at 42°C while rocking for 2 hours. The volume of prehybridization solution used should be 20 to 100 L per cm2 of the blot.

8. For the hybridization solution, just before you are ready to use it, heat-denature 7 L of your probe and 200 L of Herring sperm DNA in a 0.5 mL microfuge tube by placing the tubes in a 100°C hot block for 10 minutes. Before going into the hot block, wrap the tops of the tubes with Parafilm so the writing is not removed in the next step.

9. After the 10 minutes at 100°C, plunge the probe/Herring sperm sample into an ethanol ice slurry for fast chilling, making sure not to erase the labels written in marker.

10. Just before use, add all of the denatured probe/Herring sperm solution (~210 µL) to the hybridization solution.

11. Cut a corner of your bag and remove the prehybridization solution using a serological pipette. Add the hybridization solution to the bag using a fresh serological pipette (20-100 L per cm2) and reseal using the same techniques described in step 6. The blot should be hybridized at 42°C overnight while rocking to achieve maximal sensitivity.

Following Day: 1.14 Washing and preparing the Southern blot (Takes about 1 hour.)

**All the washes in this section need to be completed while rocking.**Decant and save the hybridization solution in an appropriate size tube. Store at 4C.

1. Wash the blot & test spot with 100 mL of 2X SSC/0.1% (w/v) SDS at room temperature (RT) for 3 minutes. Decant the SSC and repeat.

2. Wash the blot & test spot with 100 mL of 0.2X SSC/0.1 % (w/v) SDS at RT for 3 minutes. Decant the SSC and repeat.

3. Wash the blot & test spot in 100 mL of 0.16X SSC/0.1% (w/v) SDS at 50°C for 15 minutes.

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Decant the SSC and repeat.4. Rinse the blot & test spot in 100 mL of 2X SSC at room temperature for 1 minute.5. Dry the blot and test spot on large Kimwipe™ and then wrap in plastic wrap and store in

the refrigerator.6. The hybridization mixture containing the biotin-labeled probe may be reused. Store the

mixture at 4C for several days or at -20C for longer periods. Placing the hybridization solution in a boiling water bath and cooling on ice just prior to use should denature the probe.

1. 15 Development of Blot _____ HAZARDOUS CHEMICAL INFO:-NBT/BCIP is highly toxic. WEAR GLOVES when handling and dispose of all liquid waste containing NBT/BCIP in the appropriate waste container.

1. Wash your blot and test spot in Buffer 1 at room temperature for 1 minute with sufficient buffer to cover the blot. Decant Buffer 1 into the sink.

2. Incubate the blot and test spot in Buffer 2 in a plastic container for 1 hour at 65°C while rocking, with sufficient buffer to cover the blot.

3. Decant Buffer 2, place the blot and test spot on a Kimwipe™.4. Dry the blot and test spot at 80oC for 15 min.5. Wash the blot and test spot in freshly made streptavidin alkaline phosphatase (SA-AP)

conjugate for 25 minutes at room temperature. 1 µL SA-AP per 1 mL Buffer 2. (Add only enough SA-AP conjugate to cover the blots, ~10 mL. Use gentle agitation and occasionally pipette SA-AP over the blots.)

6. Decant and save the SA-AP in a 15 mL tube. Save for step #9.7. Wash the blot and test spot in Buffer 1 using 20 to 40-fold greater volume than

employed in step 1. Gently agitate blot for 15 minutes in Buffer 1.(If you used 10 mL diluted SA-AP conjugate in step 1, wash with at least 200-400 mL Buffer 1.) Decant Buffer 1 into the sink.

8. Wash the blot and test spot for 10 minutes in Buffer 3 while rocking. Decant Buffer 3 into sink.

*Do steps 9 and 10 at the same time and monitor the rate of color development. The tube of saved SA-AP acts as a positive control.** When you add the NBT/BCIP in the next steps, be sure to start a timer and note down when color changes occurred.

9. Add 1 mL NBT/BCIP solution to the saved SA-AP. A blue color should develop overtime. Wear gloves when working with NBT-BCIP.

10. Add 9 mL of NBT/BCIP solution to the blot and test spot. Allow the blot and test spot to develop for 15 minutes to 1 hour. Agitate the Tupperware during development.

11. DNA bands will be most evident on only one side of the blot (check your blot for correct orientation). Check your blot every 2 minutes to ensure that over-development does not occur.

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12. Once bands have developed, decant the NBT/BCIP solution in the appropriate waste container and wash the blot in TE buffer. This will terminate the color development reaction. The TE can then be decanted into the sink.

13. Record the amount of time development took in NBT/BCIP for the Southern blot and the test spot.

14. Let the blots dry on a large Kimwipe™. Once dry, wrap in plastic wrap and label. The lab tech will image and distribute the blots to you for your notebooks.

15. Measure the image of the cracking gel and compare the relative position of the plasmid band to the results of the blot. Interpret your results.

1.16 Designing Primers __________

Using the nucleotide sequence provided, you will design gene-specific primers to use in polymerase chain reaction (PCR). In order to exponentially amplify a unit product that includes all or part of the coding region for the glutathione S-transferase (GST) gene, you should design two primers (Forward and Reverse). One primer should recognize part of the GST sequence and the other primer should recognize part of the plasmid sequence. This will allow for the amplification of only the GST plasmid and in no way will endogenous GST be amplified. The forward primer sequence can be taken directly from the 5’ 3’ nucleotide sequence provided. The forward primer will bind, using Watson-Crick base pairing, to the 3’5’ complementary strand. (The complementary strand is not shown with the sequence, but is included in the illustration on the next page.) The forward primer will bind the 3’5’ strand in the 5’3’ orientation (see illustration on the next page). The reverse primer needs to be the reverse compliment of the sequence provided, therefore allowing for Watson-Crick base pairing to the 5’3’ sequence we give you. The reverse primer will bind at the 3’ end of the targeted sequence in the 5’3’ orientation (see illustration).

Please use the illustration below and go through the exercises that follow to help you understand. (Remember, DNA polymerase III, which we will use for PCR, must be primed and will produce a complimentary strand in the 5’3’ direction.) Please make sure your final primers are written in the 5’3’ orientation when you submit them.

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Guidelines for Designing Primers:

1. The primers should be 18-25 nucleotides in length with a calculated TM of approximately 50-55oC.

2. TM= 2°C (A&T) + 4°C (G&C)3. Primers should start and end with G’s or C’s4. The nucleotide composition of the primer should be ~50% G/C. 5. Try to avoid long stretches of the same nucleotides (i.e. more than 4 of a particular

nucleotide in a row).6. Make sure to avoid large amounts of complementarity between the Forward and

Reverse primer and between the 5’ and 3’ ends of each primer.

Example of Reverse primer design

1) 5’-GTCGTACGTACGGCGTCGTCC-3’ This is the sequence you want the reverse primer to bind to.

2) 3’-CCTGCTGCGGCATGCATGCTG-5’ This is the sequence written backward; now it is 3’5’.

3) 5’-GGACGACGCCGTACGTACGAC-3’ These are the complementary bases of the sequence above. This is the primer sequence, 5’3’ that will bind to the sequence above. It is the reverse primer you would order from the company.

Now you try:Here is a portion of the GST gene sequence that you want to use as a reverse primer

site. Design the primer that will bind to this sequence:

5’-GATATTAGATACGGTGTTTC-3’

Please draw on the DNA diagram below where your forward and reverse primers will bind and which direction the Taq DNA polymerase will transcribe.

5’-------------------------------------------------------------------------------------------------------3’

3’-------------------------------------------------------------------------------------------------------5’

Part of the plasmid and the glutathione S-transferase (GST) nucleotide sequence (bold and underlined) is shown below:

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5

’GTGGGGAAGGTGAGCGGATACAATTTCACACGGAAACAGTATTCATGTCCCCTATACTAGGTTATTGGAA

AATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGA

GCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGG

AGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATA

CGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTC

AATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAG

ACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAG

ATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTA

TGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTT

TGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTAT

ATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCG

GATCTGGTTCCGCGTGGATCTCGTCGTGCATCTGTTGGATCCCCGGGAATTCATCGTGACTGACTGACGATCTG

CCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAA

GCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGA

CCCAGTCACGTAGCGATAGGCGGAGTGTATAATTCTTGAAAAACAAAAGGGCCTCGGGAAACGCCTATTTTTATA

GGTTAATGGCATGAAAATAAATGGTTTTCTAAAACGTCAGGGGGGCACTTTTTCGGGGAAAAGGTGGCGCGGAA

ACCCCTTATTTTGGTTTATTTTTTTTCTAAAAAAACATTTTCAAAATAATGTTATCCCCCCCCTCATGGAAAAAAAAA

ATAAAACCCCCGGGGAAAAAAAAAGGGGCTTTTTCAAAAATAAAAAAAAATATTTTGTAAAAAAAAAAAAAGGG

GGGAGAGAA-3’

One or both of your primers should be from outside the GST sequence (bold and underlined), meaning it is a part of the plasmid. Once you have designed the primers, fill out the oligonucleotide request form. The primers will then be made on a DNA synthesizer.

The glutathione S-transferase protein consists of 232 amino acids. The sequence, using the one-letter abbreviation for each amino acid, is shown below.

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPGIHRD*

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Day Before 1.17 _____________________________________________________ 1. Using the streaking method, plate fresh transformed and non-transformed bacteria

from your old plates (1.6).

1. 17 PCR 1. Set up seven 0.5 mL PCR reaction tubes according to the PCR chart in Appendix K. Read

the chart carefully and make sure you add the correct amounts of reagents. PCR is a very sensitive reaction and adding the incorrect amounts of reagents may cause poor results. Appropriately label your tubes with your group color and tube number!

2. For Sample 4, pick up three individual colonies from your transformed plate with a sterile toothpick and place into a 0.5 mL microfuge tube containing 50 µL of sterile dH2O. Do the same for Sample 5 using your non-transformed bacteria. Lyse the bacteria at 99°C for 5 min in the Thermocycler. Use 3 µL of this bacterial solution as your “template DNA” for your sample 4 and 5, respectively.

3. Before mixing the reagents, calculate how much water must be added to make a total of 25 L (including the Taq Polymerase). This is necessary because the amount of template DNA or MgCl2 that you add may differ from tube to tube.

4. Be sure each reagent is completely thawed and well mixed before removing any. It is also a good idea to mix the tube gently by flicking it and spinning it down in the bench top spinner.

5. Be sure to change your pipette tip each time you pipette something .6. If you add too much DNA, nonspecific amplification may occur—check with your TA to

make sure you don’t need to adjust your DNA volume based on the concentration of your plasmid.

7. Add all reagents while the tubes are on ice.8. The last reagent you should add is the Taq polymerase to each tube.9. Once all reagents are added to the tubes, mix by flicking the tubes and spin them briefly

to bring all the liquid to the bottom of the tube. Note: Only spin tubes briefly (5 sec.), 0.5 mL tubes are thin-walled and can crack if microfuged for too long.

10. Keep the tubes on ice until the entire class is ready to load the thermocycler.11. The thermocycler will run for approximately 3 hr. After the 3 hr period, the

thermocycler will stay at a constant 4C until the tubes can be placed at -20°C by the lab technician or TA. This will ensure that the PCR products do not degrade.

1.18 Examining the PCR product _____ HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

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1. Prepare a 2% agarose gel. Note: The 2% agarose solution will solidify quickly! Pour gel while still relatively hot.

2. Remove 20 µL of PCR product from each tube; add to 4 µL of 6X DNA loading buffer. Store the remaining PCR product at 4°C. (**add 5 ul of 6X to the PCR tube**)

3. Once your gel is set, remove the comb and place the gel in the running box. Cover the gel with 1X TAE buffer.

4. Load your DNA samples and 10 µL of 100 bp ladder into the gel. 5. Add 20 µL of 1X DNA loading buffer to any empty lanes.6. Run gel at 100 volts for 1.5 hours.7. Once the electrophoresis is complete, stain your gel for ~20 minutes in ethidium

bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.8. Destain, examine, and photograph gel.

Be sure to discuss your results with Dr. V before you leave.

1.19 Searches of the sequence using BLAST __________ ( B asic L ocal A lignment S earch T ool) The plasmid DNA has been sequenced. You will receive a printout of the results. You will analyze this information using a computer program called BLAST.

To access the program, go to http://www.ncbi.nlm.nih.gov/BLAST

BLAST1. Open the internet browser and go to http://www.ncbi.nlm.nih.gov/pubmed/

2. Scroll down the page and select BLAST from the popular searches menu.

3. Select nucleotide BLAST and copy the entire GST nucleotide GST sequence (provided to you via e-mail) into the query sequence box.

4. Make sure the database that is selected is nucleotide collection (nr/nt).

5. Click on the BLAST button at the bottom of the page and wait until the search is completed (results will be presented in a new tab).

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Module 2Proteomics

Introduction In this module, you will compare the proteins found in fractions of Paramecium cilia. You will run a one dimensional polyacrylamide gel, remove and cut into bands the lanes from the membrane and axoneme protein samples, then compare the proteins present in those samples using mass spectrometry.

Before starting this module, you should read extensively about the structure of cilia. Look for which proteins are in the ciliary membrane vs the axoneme. You will be asked to make predictions about which proteins will be in each ciliary fraction and their relative abundance.

2.0 Practice Preparation of Resolving Gel HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container.

1. Obtain one 1.5mm spacer glass plate and one short glass plate; wipe with methanol and a Kimwipe™ until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

2. Assemble the gel casting apparatus (see Appendix N). Assemble on a flat surface and then clamp shut.

3. Insert the 1.5mm comb and use a Sharpie to draw a line across the glass 0.7 cm below the bottom of the wells of the comb. Once the line is drawn, remove the comb.

4. Test to see if the apparatus is leak-proof by squirting some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Make sure to remove the water before pouring the gel. Use a piece of Whatman filter paper to remove water droplets from between the plates of glass.

Before you pour your gel, have the TA or lab tech check your apparatus!

5. Prepare a 12% resolving gel in a 15 mL tube according to the following directions. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL.

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Sterile dH2O 3.29 mL

4X Resolving Buffer pH 8.9 2.60 mL 30% Acrylamide stock 4.00 mL TEMED 10 L Last: Fresh 10% Ammonium persulfate 100 L

6. Gently invert the solution to mix WELL. 7. Using a serological pipet, quickly pipette the acrylamide mixture into the cassette by

allowing the solution to run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

8. Overlay the acrylamide immediately with dH2O using a P-1000. Do this by gently adding the dH2O. You will be able to see a distinct line between the dH2O and the resolving gel solution.

9. Allow the gel to polymerize for AT LEAST 30 minutes. Any extra acrylamide mix in your tube will be a good gauge for polymerization. (Make sure the cap is on.)

10. After your gel has polymerized, remove the water and check the interface – it MUST be flat. If it is not, the gel must be poured again.

11. Once your gel has polymerized and has a flat interface, cover completely with water and wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

2.00 Isolation of Cilia (Done for you) _____ Your TA will prepare the cilia from Paramecium using the following protocol. Approximately 6 L of wild type cells and 9-12 L of mutant cells are needed. The cells are not all harvested on the same day. Below is the protocol for harvesting the Paramecium cells, typically 3 L at a time. (All solution recipes are shown in Appendix A.):

1. Filter Paramecium cell culture by pouring culture through a funnel lined with cheese cloth sandwiched between 2 large Kimwipes™ into a clean 2800 mL flask.

2. Pour the culture through and IEC Clinical continuous flow centrifuge to concentrate the cells into ~300 mL.

3. Concentrate the cells further using an IEC HN-SII clinical centrifuge and pear-shaped tubes. Cells are spun for 2 minutes at ¾ speed (~500 x g) and collected using a Pasteur pipette.

4. Pipette cells into a beaker containing 200 mL of room temperature Dryl’s solution. Centrifuge again in pear-shaped tubed for 2 minutes at ¾ speed. Remove trichocysts (fluffy layer on top of pellet) and put in waste container. Transfer cells to 200 mL fresh Dryl’s solution. Mix and centrifuge again. The cells are washed a total of 3 times.

5. After the final wash, add cells to a flask containing 40 mL cold Dryl’s solution, add 40 mL cold STEN buffer. Keep on ice for 10 minutes to immobilize the cells.

6. Add 16 mL Cilia Shock Buffer to cells, swirl the beaker and place on ice for 2-5 minutes. During this time the cells will deciliate – examine under inverted microscope to watch

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the progress of deciliation and to make sure that the cell lysis is minimal (preferable <1%). If cells begin to lyse, spin down immediately.

7. Transfer cell solution to pear-shaped flasks and centrifuge for 2 minutes at full speed (~850 x g). Pour the supernatant into 2 clean pear-shaped tubes, leaving cell bodies in the old tubes. Spin supernatant again for 2 minutes at full speed.

8. Transfer supernatant to 30 mL Corex tubes with rubber sleeves. Spin in Beckman J2-21 Centrifuge (JA-17 rotor) at 14,500 rpm for 20 minutes.

9. Add 3 mL of 10 mM Tris EDTA pH 8.3 to one tube and vortex for 5 minutes. Transfer the resuspension to the next tube and vortex for 5 minutes. Continue to consolidate the pellets until all pellets have been resuspened.

10. Rinse the empty tubes with 2 ml Tris-EDTA pH 8.3 to collect any residual cilia. The total volume of the sample should now be ~5 mL.

11. Place the resuspended cilia in a fresh 15 mL Corex tube and wash the last tube with 1-2 mL Tris EDTA pH 8.3.

12. Spin at 19,500 rpm in JA-20 rotor for 30 minutes.13. Pour off the supernatant. Resuspend the pellet in 400 - 600 µL of 10 mM Tris (pH 8.0).

Be sure the suspension is homogenous. Store at -80°C for future use.

2.1 Ultracentrifugation_________________________________________Each group will be provided with a cilia sample to subfractionate. Keep these on ice.

1. You are provided with 66% sucrose solution. In three 15 mL tubes, create three concentrations of sucrose (55%, 45% and 20%) using the calculations in Appendix L.

2. Observe the TA making a step gradient of sucrose. Label your ultracentrifuge tube with your group color.

3. Next, create the sucrose gradient by first adding 0.7 mL of 66% sucrose to the Ultraclear Ultracentrifuge tube, then 1.4 mL 55% sucrose, then 1.4 mL 45% sucrose and finally 0.7 mL 20% sucrose.

4. Carefully place 200 to 300 µL of your protein sample on top of the sucrose gradient. Rinse the empty protein sample tube with 100 µL of 10 mM Tris (pH 8.0) + protease inhibitors and add to the gradient. Make sure your Ultracentrifuge tube is labeled for your pair using a Sharpie. Balance your pair’s tube against another pair’s before placing the ultracentrifuge tubes in their buckets using 10 mM Tris (pH 8.0) + protease inhibitors. Note the number on the bucket that corresponds to your sample.

5. Your TA will set up and run the ultracentrifuge.

6. The Beckman L8-80M Ultracentrifuge (Sw60Ti rotor) will spin at 45,000 rpm at 4°C for 1.5 hours.

7. After centrifugation, three sample interfaces should be visible (see Appendix M). We are going to collect the ciliary membrane which is at the interface between the 20% and 45% sucrose layers and axonemes which are at the interface between 55% and 66% sucrose.

29

8. Use an insulin syringe to poke a hole in the side of the ultracentrifuge tube and suck out the white, cloudy interface between the 20% and 45% sucrose layers. Place the collected sample into a fresh 15 mL Corex tube.

9. Repeat the process for the layers at the 55% - 66% interface.10. Add 12 mL of 10 mM Tris pH 8.0 + protease inhibitors to each tube. Cover the tubes

with Parafilm and invert numerous times to mix well. 11. Balance the tubes in their rubber sleeves. Make sure the tubes are labeled with your

type of sample and group. 12. Centrifuge in the Beckman J2-21 (JA- 17 rotor) at 17,000 rpm at 4°C for 30 minutes.13. Immediately remove the samples, pour off the supernatant and discard in the waste

container. Allow tubes to rest upside down on a Kimwipe™ for a minute and then place on ice.

14. Resuspend the pellets in 50 µL of Membrane Buffer with protease inhibitors. While resuspending the pellets, try to keep the tubes on ice.

15. Move the samples to a 0.5 mL tube that is well labeled. Rinse the Corex tubes with an additional 25 – 50 µL of membrane buffer + protease inhibitors to remove all the sample and combine this with your original sample. Mix well.

16. Remove 16 µL from each of your samples. Again, make sure the samples were well mixed. Place the 16 µL in a labeled microfuge tube for the Pierce BCA Protein Assay.

17. Store both tubes (samples) at -80°C.

2.2 Preparation of Resolving Gel HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container.

12. Obtain one 1.5mm spacer glass plate and one short glass plate; wipe with methanol and a Kimwipe™ until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

13. Assemble the gel casting apparatus (see Appendix N). Assemble on a flat surface and then clamp shut.

14. Insert the 1.5mm comb and use a Sharpie to draw a line across the glass 0.7 cm below the bottom of the wells of the comb. Once the line is drawn, remove the comb.

15. Test to see if the apparatus is leak-proof by squirting some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Make

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sure to remove the water before pouring the gel. Use a piece of Whatman filter paper to remove water droplets from between the plates of glass.

Before you pour your gel, have the TA or lab tech check your apparatus!

16. Prepare a 12% resolving gel in a 15 mL tube according to the following directions. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL.

Sterile dH2O 3.29 mL 4X Resolving Buffer pH 8.9 2.60 mL

30% Acrylamide stock 4.00 mL TEMED 10 L Last: Fresh 10% Ammonium persulfate 100 L

17. Gently invert the solution to mix WELL. 18. Using a serological pipet, quickly pipette the acrylamide mixture into the cassette by

allowing the solution to run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

19. Overlay the acrylamide immediately with dH2O using a P-1000. Do this by gently adding the dH2O. You will be able to see a distinct line between the dH2O and the resolving gel solution.

20. Allow the gel to polymerize for AT LEAST 30 minutes. Any extra acrylamide mix in your tube will be a good gauge for polymerization. (Make sure the cap is on.)

21. After your gel has polymerized, remove the water and check the interface – it MUST be flat. If it is not, the gel must be poured again.

22. Once your gel has polymerized and has a flat interface, cover completely with water and wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

2.3 Pierce BCA Protein Assay Pierce BCA Protein Assay (See Appendix O)Use the chart provided to develop a standard curve using BSA standards and to determine protein concentration.

1. Dilute protein samples: Make two 1:50 and 1:20 dilutions of each of your protein samples. For example, to make the 1:20 dilution, add 5 L of your sample to 95 L of sterile dH2O. For the 1:50 dilution add 2 L of your sample to 98 L of sterile dH2O. The dilutions should be done twice, not one large dilution that you split in half.

2. For your standards, add BSA and dH2O according to the directions in Appendix O. These should also be done in duplicate. This set up will provide two identical reactions.

3. Make dye solution when all your standards and unknowns are prepared; not before!: Use Solutions A and B from the Pierce Protein Assay Kit; Solutions A and B should be

31

mixed 50:1. Show the amount of dye you plan to make to the TA or technician. Be sure to plan on a few extra tubes.

4. Add 2 mL of the dye to each one of your samples and standards.5. Incubate at 37°C for 30 minutes. 6. Get OD values for standards and samples: TA will assist in the operation of the spec.

Obtain OD values for one set of standards and unknowns, then repeat the process for the second set of standards and unknowns.

a. Place your standard into a clean cuvette to blank the spec. To clean the cuvette, rinse with dH2O. Make sure to dry the outside of the cuvette with a Kimwipe™. Handle the cuvette only on the frosted sides.

b. Read OD at 562 nm. c. Obtain readings for the first set of standards and unknowns. d. Re-blank the spec with the second blank for the second set of standards and

unknowns. Obtain the OD’s for the second set. If only 1 cuvette is available, make sure to rinse with dH2O between each standard.

7. Find the average of each standard and sample duplicates.8. Establish a standard curve using the OD values obtained with your BSA standards:

graph Concentration (x-axis) vs. OD (y-axis) on excel. Using this graph and the line equation, calculate the protein concentrations in your two samples.

Do not force the line through 0.See Appendix Q for assistance.

9. E-mail your graph and calculated unknown concentrations to Ashik. Be sure to put the graph, OD values, and calculated concentrations in your lab notebook.

10. You will need your calculated concentrations of your protein sample for the next class.

2.4 SDS-PAGE HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container. -Coomassie Blue Stain and Coomassie Blue Destain are highly flammable and irritating to the skin. WEAR GLOVES when handling and dispose of in appropriate waste container.

1. Remove the resolving gel from the 4o fridge and pour a 4% stacking gel. 2. Make sure you have cleaned your comb with ethanol.3. Mix the following components in a 15 mL tube.

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Sterile dH2O 6.10 mL 4X Stacking Buffer pH 6.8 2.50 mL

30% Acrylamide stock 1.30 mL TEMED 10 L

Last: Fresh 10% Ammonium persulfate 50 L

4. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and remove any residual water using a piece of Whatman filter paper.

5. Add the ammonium persulfate to your tube. Mix gently.6. Pour the stacking gel as you did the resolving gel all the way to the top of the small glass

plate using a serological pipet. Take caution when inserting the comb into the gel, it may spurt out. If the acrylamide mix overflows when inserting the comb, it’s okay. Make sure there are no air bubbles in between the wells or at the bottom of the teeth of the comb.

This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

7. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while waiting.

8. Once the gels are set, remove them from the casting stand and assemble in the gel box (See Appendix P). Note: Do not remove the comb yet.

9. Add 1X PAGE Running buffer to the upper chamber. The buffer level should be half way between the top of the big and small glass plates.

10. Add 1X PAGE Running buffer to the lower chamber until the appropriate level for the number of gels in the box is reached.

11. Carefully remove the comb.Gel Set UpYour TA will help you determine how much sample you are going to load based on the concentration of your protein sample. Groups will share proteins samples. Each gel will have equal concentrations of the control and mutant pure ciliary membrane samples and a pre-stained protein marker (see Appendix I). Your TA will help you to equalize the concentrations of membrane and axoneme samples; these must be approximately equal in order for the samples to be comparable. Any empty lanes should be loaded with 1X SDS sample buffer.

1. Calculate the volumes of membrane or axoneme sample, 10X SDS sample buffer, and Membrane buffer needed for each tube:

a. Before using the 10X SDS Sample Buffer, add 50 µL of β-ME to 500 µL of 10X SDS Sample Buffer.

Be sure to do this in the fume hood.b. Calculate the volume of your protein that provides *60 µg. The remaining

volume should be made up with 10X SDS sample buffer and membrane buffer so the final volume should be 50 µL:

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*60 µg protein _____________ µLMembrane Buffer _____________ µL10X SDS sample Buffer 5 µL

c. Obtain the other sample (membrane or axoneme) from another group – we will help match you all up. Double check the other group’s concentration calculation so we are sure everything is correct.

*We reserve the right to alter concentrations or final volumes based on the concentrations of proteins obtained by the class. If a large amount of protein is obtained, we may increase the concentrations to 100 µg. If not enough protein is collected, we may decrease the concentration and/or loading volume.

2. Add the calculated volumes of membrane buffer, protein, and prepared 10X SDS sample buffer to a 1.5 mL tube. Place ANY remaining protein samples in the freezer.

3. Make sure your samples are well labeled.4. Boil samples for 5 minutes right before you are ready to load. After boiling, keep

samples on ice while loading gel. Be sure to mix the samples well before loading, but gently, as they will foam.

5. Using gel loading tips, load your samples in the following order: Lane 1, 3, 4, 6, 7, 9 and 10: 1X SDS sample buffer Lane 2: 20 µL Prestained protein markerLane 5: 60 µg ciliary membrane protein Lane 8: 60 µg axoneme protein

6. Run the gel at 150V (2 gels = 150V) for 1-1 ½ hours; until the dye runs off the bottom edge of the gel.

7. Remove gel carefully from the gel apparatus. Use a razor blade to cut the stacking gel portion away. Dispose of the stacking gel in the appropriate waste container.

8. Place the resolving gel into a plastic container and cover with Coomassie stain. Stain while rocking gently overnight.

Next Day (Done for you):9. Pour off the stain into original container.10. Add destain to the gel. Rock the gel gently. 11. Discard the destain after ~1 hour and pour fresh destain over the gel. Continue to rock

at room temperature.12. Again, remove the destain and replace with fresh destain. Rock gently overnight. 13. The Lab tech will save these gels in destain until the next lab.

2.5 Cutting up the gel and trypsinizing proteins in preparation for LC-Mass SpectrometryHAZARDOUS CHEMICAL INFO:

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-Acetonitrile is poisonous. It also tends to leak from pipet tips when being measured. Always wear eye protection and gloves when handling it or transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your gloves.The TA will demonstrate:

1. Clean a large glass plate with soap and water and rinse thoroughly with sterile water. Rinse again with 95% ethanol. Let the plate dry.

2. Carefully remove the “chosen” gel from its container and place it on the glass plate. 3. Each student pair should label four 1.5 mL microcentrifuge tubes, two for cilia

membrane and two for axoneme gel slices, with appropriate identification for each gel slice. For example, M1 and M2 for gel slices from the membrane lane; Ax1 and Ax 2 from the axoneme lane. There will be the same number of gel slices for the membrane and axoneme as there are students in the class, 24 total.

4. Each gel slice will be cut using a razor blade that is cleaned with 95% ethanol before using and between cutting each band.

5. Once the gel slice is cut out of the gel, the TA will slice each band into 1 mm x 1 mm cubes and place these cubes in your labeled 1.5 mL microcentrifuge tube.

6. Add 900 µL of HPLC-grade water to each tube. Incubate at room temperature for five minutes.

7. Centrifuge at high speed for 30 seconds. Using your pipettor set to 1000 µL, carefully remove the water and discard it in the waste container provided. Use a new pipet tip for each sample. Be careful not to lose the gel cubes in this process.

Note: The following steps use acetonitrile, which is poisonous. It also tends to leak from pipet tips when being measured. Always wear eye protection and gloves when handling it or transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your gloves.

8. Add 750 µL destain solution (50 mM ammonium bicarbonate, 50% acetonitrile) to each tube. Close the tube cap and mix gently by inversion. Incubate the tubes at 37°C for 20 minutes.

9. At the end of 20 minutes inspect the gel. If it is still very blue, repeat the destain process with fresh destain reagent. To do this, briefly centrifuge the tube, carefully pipet off the liquid, add 750 µL new destain reagent and incubate for an additional 15 minutes. Make sure you put the discarded destain solution in the provided waste receptacle. Repeat one more time if needed. Change gloves if you get acetonitrile on them and wash skin with water if it contacts the acetonitrile.

10. When the gel sample is clear and no longer contains blue color, centrifuge the tube at high speed for 30 seconds. Carefully remove all the destain solution and discard it in the waste receptacle.

11. Add 100 µL of 100% acetonitrile to each tube. (The gel pieces should be entirely immersed, if not add more.) The gel pieces will turn white as they dehydrate. Incubate the tubes for 5 minutes at room temperature.

35

12. Centrifuge for 30 seconds and carefully pipet off all the acetonitrile. Discard in the provided waste receptacle. Use a 200 µL pipettor to carefully remove the residual liquid from the tube. If gel cubes are not completely opaque, repeat the process by adding fresh acetonitrile and incubating for another 5 minutes. Also repeat the centrifugation and removal step before moving forward.

13. Open lids and place tubes in Speed Vac for 5 minutes to dry samples completely. Close the lids before proceeding to the next step.

14. Place the tubes on ice for 5 minutes. Add 30 µL ice cold Trypsin digestion buffer solution to each tube. Incubate on ice for 5 minutes. Add more of the digestion buffer if necessary to cover gel slices. Make sure the gel slices are completely immersed in the solution.

15. Incubate the tubes on ice for an additional 30 minutes. Add more trypsin digestion buffer if necessary.

16. Transfer the tubes to a 37°C incubator. Incubate the tubes overnight (16-18 hrs).

The Next Day (Please try to begin this process around noon):17. Spin the tubes of gel slices for 1 minute at full speed (Table top centrifuge). Transfer all

of the peptide solution (supernatant) to a properly labeled 0.5 mL tube. 18. Add 150 µL of 50% Acetonitrile / 2.5% formic acid to the tubes of gel slices and vortex.

Incubate for 45 min at RT.19. Spin these tubes for 5 minutes at full speed and transfer the peptide solution

(supernatant) to their respective 0.5 mL sample tubes.20. Add 100 µL of 100% acetonitrile to the tubes of gel slices and vortex. Incubate for 20

min at RT (Repeat if needed).21. Spin for 5 minutes at full speed and transfer the peptide solution (supernatant) to the

respective 0.5 mL sample tubes. Save the tubes of gel pieces at -20o C for later extraction if needed.

22. Dry the samples using the Speed Vac for 2 to 4 hours (the TA and technician will assist with this process).

23. Your TA will bring your dried samples to the UVM Proteomics Core Facility for processing.

2.6 Mass Spec Tour the UVM Proteomics Core Facility in MLS 337 and meet to discuss how mass spec works.

2.7 Mass Spec Results Discussion_______________________________Experienced proteomics personnel will discuss how to analyze the results you receive from the Mass Spec.

2.8 Analyze results

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Your TA will help you to analyze your results from mass spec, and determine which proteins are differentially expressed.

Refer back to your predictions for the presence of proteins and their relative abundance in membrane vs axoneme. How did your predictions compare to the results?

37

Appendix A: Solutions Guide

GENERAL SOLUTIONSAmpicillin (AMP) Stock

100 mg/ml stock solution: for example, 0.5 g ampicillin sodium salt into 5 mL dH2O. Filter sterilize, and store at -20°C.

LB-AMP100 µg/mL final concentration of AMP: a 1:1000 dilution of ampicillin stock into LB broth (i.e. add 1 mL ampicillin stock (100 mg/mL) into 1 L LB Agar broth). *NOTE: Ampicillin is heat-sensitive. LB agar broth must be cooled to 60°C after coming out of the autoclave before the AMP is added. Setting a large water bath to 60°C and letting the LB agar broth cool in the water after autoclaving, for an hour, is a good way to ensure the LB agar doesn’t solidify and that it won’t be too hot for the AMP.

TE buffer – 1 LNeed (final conc.): 10 mM Tris-Cl, 1 mM EDTAMake from liquid stocks of Tris-Cl and EDTA5 mL 2 M Tris-Cl (pH 7.5)2 mL 0.5 M EDTA (pH 8)993 mL dH2O

2 M Tris-Cl (pH 8.0) – 1 L177.6 g Tris-Cl10.6 g Tris-baseIn ~950mL sterile dH2O**pH 8.0**Bring up to 1 L with sterile dH2O

0.5 M EDTA (pH 8) – 100 mL18.6 g EDTA disodium salt (FW= 372.2)In ~75 mL sterile dH2OHeat in microwave to dissolve salt***bring pH to 8.0***Bring up to 100 mL with sterile dH2O

50X TAE stock (pH 8.5) – 1 L242 g Tris Base (FW= 121.14)In ~700ml sterile dH2OCarefully add 57.1 mL Glacial Acetic Acid100 mL 0.5 M EDTA (pH 8.0)Bring up to 1 L with sterile dH2O

pH 8.5, but no adjustment needed

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6X DNA loading buffer – 100 mL0.25 g Bromophenol Blue40 g Sucrose100 mL dH2O

λ Hind III markerWant a final concentration of 100 ng/µLMake: 96 µL λ Hind III marker (Invitrogen stock)

320 µL TE68 µL 6X DNA loading buffer

20X SSC (pH 7) – 1 L175.3 g NaCl88.25 g Na3 Citrate•2H2O1 L dH2O**pH 7.0**

2X SSC – 100 mL10 mL 20X SSC90 mL dH2O

10X TBS (pH 7.4) – 1 L 25.1 g Tris HCl4.8 g Tris Base80 g NaCl 800 mL of H2O.Bring up to 1 L with high purity distilled or deionized water.

Once prepared, TBS is stable at 4°C for 3 months.

10X TBS, 1% Tween-2010 mL Tween-20 (use large orifice tips or slow serological pipetting to pick up Tween)1 L 10X TBS

Phosphate Buffer Saline (PBS) – 500 mL4.0 g NaCl0.1 g KCl0.72 g Na2HPO4

0.12 g KH2PO4

In ~400ml sterile dH2O**pH 7.4**Bring to 500 mL with sterile dH2O

39

MODULE 1

1.1: Isolation of plasmid DNA

Solution I0.50 g D-Glucose0.625 mL 2M Tris-Cl (pH 8)1 mL 0.5M EDTAAdd dH2O to make total volume 50 mL**add 5 mg/ml lysozyme just before use**

Solution II2 mL 1M NaOH1 mL 10% SDS7 mL dH2O**Prep fresh**

5M Potassium Acetate (100 mL)29.5 mL glacial Acetic Acid Bring volume close to 100 mL using dH2O Add KOH pellets until pH=4.8Bring to volume and store at 4°C.

Heat-treated RNase A (100mg/ml)Dissolve 100 mg (0.1 g) of pancreatic RNase A in 1 mL 10 mM Tris-Cl/15 mM NaCl. Store at -20 °C. Before use, heat RNase A in 100°C heat block for 15 minutes and allow tubes to cool slowly to RT.

10mM Tris-Cl/15mM NaCl0.1576 g Tris-Cl0.0876 g NaCl100 mL dH2O

Salt Saturated (SS) PhenolTris buffered Phenol pH 6.6/7.98-Hydroxyquionoline added until dark yellow/orange color

Chloroform:Isoamyl Alcohol (24:1)480 mL Chloroform20 mL Isoamyl Alcohol

TE bufferSee general solutions section

40

1.4: Transformation

50 mM CaCl2

0.73 g CaCl2

100 mL dH2O** Make fresh**

Transformation buffer1 mL 100 mM CaCl2

1 mL 100 mM Tris (pH 8.0)1 mL 100 mM NaCl7 mL dH2O**Store at 4°C**

1.6: Secondary selection of transformed bacteria

0.145 M Sterile Saline (pH ~7)4.25 g NaCl500 mL dH2O** Autoclave to sterilize**

1.8: Cracking gel

Cracking Buffer (pH 6.8) 0.788 g Tris-Cl1.0 g SDS0.058 g Na2EDTA•2H2O13.6 g Sucrose0.1 g Bromophenol Blue100 mL dH2O** pH 6.8**

1.9: Biotin labeling of DNA

3 M Sodium Acetate (pH 4.8)24.6 g Sodium Acetate100 mL dH2O** pH 4.8**

41

1.11: Southern blotCracking Buffer

See 1.8 above

0.5 M NaOH/0.8 M NaCl20 g NaOH46.75 g NaCl1 L dH2O

0.5 M Tris/1.5 M NaCl (pH 7)250 mL 2 M Tris-base solution (pH 8)87.6 g NaCl750 mL dH2O** pH 7**

10X SSCSee general solutions for 20X SSC

1.13: Hybridization of Southern Blot

Prehybridization Solution (per group, prep fresh)5 mL Formamide2.5 mL 20X SSC0.5 mL 100X Denhardt’s solution (doesn’t keep more than 24 hours!)0.25 mL 1 M Phosphate Buffer200 µL Herring sperm DNA (2mg/mL, made fresh), freshly denatured

Hybridization Solution (per group, prep fresh)4.5 mL Formamide 2.5 mL 20X SSC0.1 mL 100X Denhardt’s solution (doesn’t keep more than 24 hours!)0.4 mL 1 M Phosphate Buffer1.5 mL dH2O200 µL Herring sperm DNA (2mg/mL), freshly denaturedBiotin-labeled probe DNA

100X Denhardt’s Solution0.2 g Ficoll 0.2 g Polyvinylpyrrolidone0.2 g Bovine Serum Albumin (BSA)10 mL sterile dH2O**Doesn’t keep more than 24 hours**

42

1.14: Detection of DNA

2X SSC/0.1% (w/v) SDS 50 mL 20X SSC450 mL dH2O0.5 g SDS

0.2X SSC/0.1% (w/v) SDS 5 mL 20X SSC495 mL dH2O0.5 g SDS

0.16X SSC/0.1% (w/v) SDS 4 mL 20X SSC496 mL dH2O0.5 g SDS

1.15: Development of BlotBuffer 1: Final Concentration: 0.1 M Tris-Cl, 0.15 M NaCl

8.7 g NaCl15.76 g Tris-ClBring to 1 L with dH2O

Buffer 2: 3% (w/v) BSA in Buffer 13 g BSA per 100 mL Buffer 1**Doesn’t keep more than 24 hours, prep fresh**

SA-AP: **Needs to be made immediately before use**1 µL SA-AP per 1 mL Buffer 2 (approx. 10 mL needed per group)

Buffer 3Final concentration: 0.1 M Tris-Cl pH=9.5, 0.1 M NaCl, 50 mM MgCl2

15.764 g Tris-Cl5.844 g NaCl10.15 g MgCl2

Bring to 1 L with dH2O

1.17: PCR20 µM Primer Dilutions (from 500 µM stock primers)

5 µL 500 µM stock120µL dH2O**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller batches**

43

10 mM dNTP mix (from 100 mM individual dNTP stocks)500 µL dCTP500 µL dTTP500 µL dATP500 µL dGTP3 mL dH2O *1 µL of 10 mM dNTP mix per 50µL reaction*

MODULE 2

3.0 Preparation of Cilia

Dryl’s solution – 1 L (final concentrations)1 mM Na2HPO4

1 mM Na2H2PO4

2 mM Sodium Citrate1.5mM CaCl2

pH 6.8

STEN Buffer – 100 mL17.12 g Sucrose0.316 g TrisCl0.068 g Na2EDTA0.036 g NaCl100 mL dH2O*pH to 7.5 and fill**store at 4C

Cilia Shock Buffer (final concentrations)180 mM KCl 60 mM CaCl2

Membrane Buffer – 50 mL0.331 g Tris-Cl0.048 g Tris base0.186 g KCl0.051 g MgCl2•6H2O0.019 g EGTA50 mL dH2O*pH to 7.4***add 1% Triton X-100 before use

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LeupeptinDissolve in dH2O 1 mg/mLStore at -20C in 1 mL aliquots*Stable for 6 months*

Pepstatin A Dissolve in methanol 1 mg/mL(5 mL methanol into 5 mg bottle and shake)Store at -20C in 1 mL aliquots*Stable for 1 month*

100 mM PMSF stock (Phenylmethylsulfonyl fluoride)0.871 g PMSF50 mL 100% ethanolAdd ~1.75 g of molecular sieves to each bottle to absorb any water*inactive in 30 minutes @ 4C in aqueous solution*

Preparation of Membrane Buffer with Protease Inhibitors1 mL Membrane Buffer2 µL Pepstatin2 µL Leupeptin10 µL PMSF2 µL IAA (0.02 g/mL)*Add these immediately before use*

Tris EDTA – 100 mL0.0121 g Tris Base0.0034 g Na2EDTA100 mL dH2O*pH 8.3*

3.1 Ultracentrifugation

10 mM Tris pH 8.05 mL 1M Tris, pH 8.0495 mL dH2O

66% Sucrose171 g Sucrose90 mL 10 mM Tris pH 8.0Make the night before and place in shaker at 37o C overnight to dissolve

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3.2 Preparation of Resolving Gel

4X Resolving Buffer (pH 8.9) – 100 mL18.17 g Tris base10 g SDS (measure in hood)100 mL dH2OAdjust pH to 8.9 using conc. HCl

3.4 SDS-PAGE

4X Stacking Buffer (pH 6.8) – 100 mL6.055 g Tris base0.4 g SDS100 mL dH2OAdjust pH to 6.8 using conc. HCl

10X SDS Sample Buffer – 25 mL15.6 mL 8X TrisCl/SDS (pH 6.8)10 mL Glycerol2.0 g SDS5 mg Bromophenol blue*Add 1 µL of β-Mercaptoethanol to 10 µL of 10X SDS Sample Buffer*

8X TrisCl/SDS (pH 6.8)a. g Tris base dissolved in 35 mL dH2OAdjust the pH to 6.8 with concentrated HCl, and bring to 50 mL volume. 0.4 g SDS

5X Running Buffer7.55 g Tris Base36 g Glycine*Bring solution to 500 mL volume and stir. pH should be 8.3 but don’t adjust.*Add 2.5 g SDS. **Store at 4°C** Dilute to 1X before using

Coomassie Blue Stain – 500 mL200 mL Methanol50 mL Glacial Acetic Acid1 g Coomassie Blue250 mL dH2O

46

Coomassie Blue Destain – 1 L200 mL Methanol 75 mL Glacial acetic Acid725 mL dH2O

3.5 Band Cutting and Trypsinization of Gel Pieces

100 mM ammonium bicarbonate stock – 250 mL1.977 g ammonium bicarbonateBring to volume with dH2O

Destain solution50 mM ammonium bicarbonate50% acetonitrile

Trypsin Digestion buffer – enough for ~26 samples42 µL Trypsin (20 µg/50 µL)560 µL 100 mM ammonium bicarbonate stock70 µL 100% acetonitrile700 µL dH20Keep on ice!

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Appendix B: Sterile Technique

It is very important in microbiology and genetics to work with pure cultures. Unfortunately, this is difficult. The world around us is covered with microorganisms. Microorganisms are even carried on dust particles in the air. In order to protect sterile broth, plates, solutions and pure cultures from the microbes all around us, we must practice sterile (aseptic) technique. This simple means that sterile surfaces or sterile media must be protected from contamination by microbes in the air or residing on non-sterile surfaces. A simple example of the problem is that a sterile petri plate can become contaminated with bacteria when the lid is removed. In sterile technique, only sterile surfaces touch other sterile surfaces and exposure to the air is kept to a minimum.

In the classroom, you often need to practice sterile technique when you inoculate a pure culture of a microorganism into fresh medium. Sometimes this is a transfer to a tube of liquid broth and at other times, it is a transfer to a petri plate-containing agar. While there are other circumstances that require sterile technique, these are the most common and they will be described in more detail on the pages that follow.

Here are a few simple techniques to help you along your way: Always close tip boxes and tube containers. Always place caps face-down on counters and replace them promptly onto bottles and

secure them. Never leave plates open to the air; only remove the lids to spread something or remove

something. Do not hold plates close to your face.

Appendix C: Spread Plate Technique

1. Dispense the appropriate volume of sample into the center of a sterile agar plate.2. Dip the glass spreader (aka “hockey stick”) in alcohol or gently spray alcohol on the

hockey stick over the sink. 3. Pass the spreader through the flame of a Bunsen burner to burn off the alcohol. (This

sterilizes the spreader.)

***IMPORTANT***Keep your hand above the spreader at all times (i.e., keep it tilted toward the floor) or

flaming alcohol may roll toward your hand.

4. Cool the spreader by touching it to the agar where there is no sample.5. Spread your sample over the entire surface of the agar.6. Sterilize the spreader before putting it back on the bench.

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Appendix D: Use of a Rainin Pipettor

Take note: Never rotate the volume adjustor beyond the upper or lower range of the pipettor, as

stated by the manufacturer. Never use the pipettor without the tip in place; this could ruin the precision piston that

measures the volume of fluid. Never lay down the pipettor with filled tip; fluid could run back into the piston. Never let plunger snap back after withdrawing or ejecting fluid; this could damage the

piston. Never immerse the barrel of the pipettor in fluid. Never flame your pipettor or pipette tips.

If you drop your pipettor, the precision piston system can be damaged; therefore, if your pipettor is dropped, be sure to check the pipetting accuracy has not been affected.

Recommended Volume Ranges:Model p10: 0.5-10 μL, the number after the decimal point is in redModel p20: 2-20 μL, the number after the decimal point is in redModel p200: 20-200 μL, there is no decimal pointModel p1000: 200-1000 μL, the numbers after the decimal point are in black

Pipetting Directions – Method1. Set the desired volume by holding the pipettor body in one hand and turning the

volume adjuster knob until the correct volume shows on the digital indicator. Approach the desired volume by dialing downward from a larger setting.

2. Press tip onto shaft by a slight twisting motion.3. Depress the plunger to FIRST POSITIVE STOP. This part of the stroke is the calibrated

volume displayed on the digital micrometer.4. Holding the pipette man vertically (never more than 20˚ from vertical), immerse the tip

just below the level of the liquid.5. Allow the pushbutton to return SLOWLY to the up position. Move the tip so that it stays

slightly below the level of the liquid as you draw up.6. Wait one to two seconds to ensure that the full volume of sample is drawn up into the

tip.7. Withdraw the tip from the sample liquid.8. To dispense the sample, place the tip end against the sidewall of the receiving vessel

and depress the plunger to the FIRST STOP. Wait one to two seconds. Then depress the plunger to the SECOND STOP, expelling any residual liquid in the tip.

9. With the plunger fully depressed, withdraw the pipettor from the vessel carefully with the tip sliding along the wall of the vessel.

10. Let the plunger return slowly to the UP position. If an air bubble is observed, re-pipette the sample.

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11. Pre-rinsing the tip with the liquid being pipetted is recommended. A significant film may be retained on the inside wall of the tip, resulting in an error. Since the film remains relatively constant in successive pipettings with the same tip, refilling the tip a second time and using this quantity as the sample may obtain good reproducible results.

12. Discard the tip by depressing the tip ejector button smartly in the appropriate waste container.

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APPENDIX E:

PIERCE PROTEIN ASSAY for Module 1 Pipetting ExerciseOD Value

Label L Alb (Stock 2 mg/mL)

L dH2O mL Dye [Alb g/mL] Series 1 Series 2

S1 0 100 2 0S2 2.5 97.5 2 50S3 5.0 95.0 2 100S4 7.5 92.5 2 150S5 10.0 90.0 2 200S6 12.5 87.5 2 250S7 15.0 85.0 2 300S8 25.0 75.0 2 500

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Label L Sample

L dH2O

mL Dye

ODValue

g/mL in Cuvette

g/mL Original Solution

Unknown 1 100 0 2Unknown 2 100 0 2

Unknown 1 100 0 2Unknown 2 100 0 2

APPENDIX F: GST Plasmid Map

Websites: www.GEHEALTHSCIENCES.comhttps://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/

1314716762536/litdoc28956795_20140411174609.pdf

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APPENDIX H: Streak Plate Method

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APPENDIX I: Frequently Used DNA/Protein Markers

Lambda DNA-Hind III Digest Prestained Protein Marker

100 bp DNA Ladder 1Kb DNA Ladder

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APPENDIX J: Southern Blot Assembly

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Platform

10 CM

10X SSC

Wick

Paper Towels

Whatman Paper

Whatman Paper

Nitrocellulose

Agarose Gel

APPENDIX K: PCR Reagents and Conditions for 1.17

Cycling Program: GST

95°C 5 min Initial Denaturation

95°C 1 min5X50°C 1 min

72°C 1 min

95°C 1 min25X51°C 1 min

72°C 1 min

72°C 10 min Final elongation4°C HOLD ***************

SAMPLESInitial Stock Concentration Components 1 2 3 4 5 6 7

Template DNA* 1 µL 1 µL 1 µL3

colonies3

colonies 1 µL 1 µL 10X Buffer 5 µL 5 µL 5 µL 5 µL 5 µL 5 µL 5 µL25 mM MgCl2 0.5 µL 1 µL 3 µL 3 µL 3 µL 3 µL 3 µL10 mM dNTPs 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL20 µM Forward Primer 1 µL 1 µL 1 µL 1 µL 1 µL XXX 1 µL20 µM Reverse Primer 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL XXX5 U/µL Taq Polymerase 0.5 µL 0.5 µL 0.5 µL 0.5 µL 0.5 µL 0.5 µL 0.5 µL dH2O

*NOTE: Samples 1-3, 6 & 7: Use PLASMID DNASample 4: Transformed ColoniesSample 5: Non-Transformed ColoniesDESIRED FINAL VOLUME: 25 µL

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APPENDIX L: Sucrose Gradient Calculations

How to make __% Sucrose from 66% Sucrose

Add __ mL of 66% sucrose

Add __ mL 10 mM Tris, pH 8.0

55% Sucrose 8.00 2.0045% Sucrose 6.35 3.6520% Sucrose 2.45 7.55

How to Create the Sucrose Gradient:

First add 0.7 mL 66% Sucrose,Then add 1.4 mL 55% Sucrose, Then add 1.4 mL 45% Sucrose,Finally add 0.7 mL 20% Sucrose.

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APPENDIX M: Sucrose Gradient Tubes after Ultracentrifugation

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APPENDIX N: Protein Gel Plate Setup

Mini-PROTEAN®

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APPENDIX O: PIERCE PROTEIN ASSAY for Module 2

Label L Sample

L dH2O

mL Dye OD Values

Average OD Value g/mL in

Cuvette

g/mL Original Solution

2222

Label Average g/mL Original Solution of each treatment

g /LOriginal solution

L of protein that equates to 60 g total

Ciliary Membrane Axoneme

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OD ValueLabel L Alb (Stock

2 mg/mL)L dH2O mL

Dye[Alb g/mL]

SeriesA

Series B

Average

S1 0 100 2 0S2 2.5 97.5 2 50S3 5.0 95.0 2 100S4 7.5 92.5 2 150S5 10.0 90.0 2 200S6 12.5 87.5 2 250S7 15.0 85.0 2 300

APPENDIX P: Protein Gel Running Setup

Mini-PROTEAN®

APPENDIX Q: Graphing your protein data in Excel

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1. Enter your standard concentrations into column A.2. Enter the measured ABS into columns B and C.3. Calculate average of each reading into column D.4. Highlight columns A and D.5. On the insert tab, choose scatter plot with no lines.6. Right click on the graph data and choose trend line.

a. Click on linearb. Choose Display equation on chartc. Choose Display R-squared value on chart

7. Label axes, and title.

APPENDIX R: RPM to G-Force Conversions

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Equipment RPM G-Force (RCF or × g)Damon IEC clinical HN S-II centrifuge 3/4 500Damon IEC clinical HN S-II centrifuge Full 850Beckman J2-21 w/JA 14 rotor (250 mL tubes) 5,000 3,840Beckman J2-21 w/JA 17 rotor 5,000 3,440Beckman J2-21 w/JA 17 rotor 9,500 12,400Beckman J2-21 w/JA 17 rotor 15,000 31,000Beckman J2-21 w/JA-17 rotor 17,000 39,000Beckman J2-21 w/JA-20 rotor 14,500 25,400Beckman J2-21 w/JA-20 rotor 19,500 46,000IEC Centra 7 Desktop w/ 15 mL tubes 2,800 1,098IEC Centra 7 Desktop w/ 50 mL tubes 2,800 1,098Eppendorf Centrifuge 5702 2,800 1,120Eppendorf Centrifuge 5702 2,800 1,180Eppendorf Centrifuge 5415 C 14,000 15,980Sorvall Legend Centrifuge 14,000 18,800Ultracentrifuge 45,000 208,000Ultracentrifuge 21,600 45,000

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