Restriction Analysis of Plasmid DNA

SESSION 1/DAY 1:RESTRICTION DIGEST REACTIONS*MOLECULAR BIOLOGY FINAL *Begin Here After Biotech PP and Electrophoresis activities

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Each restriction enzyme cuts DNA wherever its recognition site appears.Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site.

Many recognition sites are palindromes.

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BamHI…NNNGGATCCNNN… …NNNG GATCCNNN… …NNNCCTAGGNNN… …NNNCCTAG GNNN…HindIII…NNNAAGCTTNNN… …NNNA AGCTTNNN… …NNNTTCGAANNN… …NNNTTCGA ANNN…

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Before We Begin: This is a restriction Enzyme Map

The circles below represent bacterial plasmids (loops of DNAfound inside prokaryotes). The orange section is a gene for the resistance of an antibiotic (either ampicillin or kanamycin)

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A restriction map identifies where restriction sites appear along the DNA

plasmid

HindIII cuts here

BamHI cuts here

What will be different between the DNA fragments produced by cutting pAMP vs. pKAN with BamHI & HindIII?

The restriction enzymes and the location where they will cut on this particular plasmid is indicated on the map (i.e.

1120 means BamH1 will cut at the 1,120th base pair starting at “12:00”)

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Cutting with Restriction Enzymes:If you are cutting with BamH1 For Example: The number 1120

represents the # of base pairs where BamH1 will cut from12:00 noon. So… If you are also cutting with HinDIII and you want to know

the size of the piece you are cutting out take 1904bp – 1120bp = 784bp (size of what will be cut out). 4539bp - 784bp = 3755bp is size

of remaining plasmid after piece cut out.

784 bp

3755 bp

DNAs can be distinguished from each other by restriction mapping.

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1904 – 1120 = 784784 bp

3755 bp 2332 bp

1875 bp4539 – 784 = 3755

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The Sample you will get for this lab will be EITHER plasmid DNA pAMP or pKAN.

Name of plasmid

pAMP; Let’s get acquainted, shall we?

•4539 base pairs •a single replication origin

•a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of penicillin)

•a single occurrence of the sequence

5' GGATCC 3'

3' CCTAGG 5'

that is cut by the restriction enzyme BamHI

•a single occurrence of the sequence

5' AAGCTT 3'

3' TTCGAA 5'

that is cut by the restriction enzyme HindIII

Treatment of pAMP with a mixture of BamHI and HindIII produces:

•a fragment of 3755 base pairs carrying both the ampr gene and the replication origin

•a fragment of 784 base pairs

•both fragments have sticky ends19

pKAN•4207 base pairs

•a single replication origin

•a gene (kanr) conferring resistance to the antibiotic kanamycin.

•a single site cut by BamHI

•a single site cut by HindIII

Treatment of pKAN with a mixture of BamHI and HindIII produces:

•a fragment of 2332 base pairs

•a fragment of 1875 base pairs with the kanr gene (but no origin of replication)

•both fragments have sticky ends

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LAB TIME!- Using Restriction Enzymes!!!Glove Up!

Put on a pair of lab glovesS, M, L, XL availableMost hands will fit in M or L gloves.

Try those sizes first unless you have particularly small or large hands.

Made of nitrile (no latex = no allergies)

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Label a Restriction Digest TubeFrom the jar with the white screw cap, remove one 1.5ml microtube.

With a lab marker, label the lid of the microtube with your period number and the first initials of each team member- (save room to record a number)

P1TDH

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LID

This is our Goal which we will complete one step at a time:

Prepare the Restriction Digest ReactionsReaction Component Volume to Add

Your Plasmid DNA Sample (0.1µg/µl) 5µlH2O 9µl

5X Restriction Buffer 4µlBamHI + HindIII Restriction Enzyme mix 2µl

Total Volume 20µl

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Add Plasmid DNAYour team was given a sample of either pAMP or pKAN plasmid DNA in a tube labeled “DNA” and a number. BE SURE TO RECORD THIS NUMBER on your restriction digest tube lid!

From this tube, use your micropipette to measure 5μl of plasmid DNA and transfer it to your Restriction Digest tube.

At 0.1μg/μl, this 5μl contains0.5μg or 500ng of DNA.

P1TDH#3

DNA1…12 5μl

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Add Water

From the tube labeled H2O, measure 9μl of water and transfer it to your Restriction Digest tube. P1

TDH#3

H2O 9μl

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Add Restriction Reaction Buffer/Loading Dye

Enzymes require a chemical environment of the right pH and concentration of ions. The 5X restriction buffer is a

concentrated mix that provides the environment needed for the restriction enzymes to work properly.

From the tube labeled 5X RE Buffer, measure 4μl of 5x Restriction Digest Buffer and transfer it to your Restriction Digest tube.

P1TDH#3

5XRE

Buffer4μl

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Add Restriction EnzymesYou will cut your plasmid DNA with

two restriction enzymes: BamHI and HindIII.

From the tube labeled BamHI + HindIII measure 2μl of the BamHI and HindIII mix and transfer it to your Restriction Digest tube. P1

TDH#3

BamHI+

HindIII2μl

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Incubate the Restriction Digest Reaction

Close the cap on your Restriction Digest tube and place it in the heating block set at 37°C.

The restriction enzymes work best at 37°C. The reactions will incubate for one hour, then be stored in a freezer until you examine them

using gel electrophoresis.

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SESSION 2/ DAY 2:GEL ELECTROPHORESIS

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Prepare Your Samples for Loading

• Do not have to add (was added to the buffer)

• Add 4µl of the 6X Loading Dye to your restriction digest sample.

If your liquids are sticking separately to the side of the tube, flick the tube with your finger and tap the bottom gently on your lab bench, or spin briefly in microcentrifuge to collect entire sample at bottom of tube.

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Load Your Sample On The FlashGel

When called, bring to the FlashGel:

Your DNA sampleMicropipette with tip

Load 6μl of your sample into a well.

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Write your team initials or team number below the well into which you loaded your sample.

Lane1

Lane2

Lane3

Lane4

Lane5

Lane6

Lane7

Lane8

Lane9

Lane10

Lane11

Lane12

Lane13

1kb ladder

Perio

d __

__

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Run the GelA power supply provides current to the electrodes and through the buffer and gel.

The progress of migration through the gel is monitored with tracking dyes that are visible without the transilluminator.

1.2% Flash Gel

200 V

8 minutes37

ANALYSIS OF GEL RESULTS

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Restriction Mapping Can Be Used To Identify Unknown DNAs

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784 bp

3755 bp 2332 bp

1875 bp

Perio

d #1

1.2% 200V 8min

Restriction Fragment Sizes

pAMP: 3755,784

pKAN: 23321875

1 2 3 4 5 6 7 8 9 10 11 12 13Promega BenchTop

1kb Ladder

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What Questions Do YOU Have?

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