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This SummerThis Summer

Meng-fu Shih Meng-fu Shih

in Dr. Fu’s Labin Dr. Fu’s Lab

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實驗室簡介實驗室簡介 PIPI ：傅化文老師：傅化文老師 研究興趣：研究興趣：

-- -- 凝血蛋白酵素接受器在細胞中移動的分子機制 凝血蛋白酵素接受器在細胞中移動的分子機制 / Molecular mechanisms of cellular trafficking of t/ Molecular mechanisms of cellular trafficking of thrombin receptors.hrombin receptors.

---- 利用次細胞蛋白體學及功能蛋白體學研究幽門利用次細胞蛋白體學及功能蛋白體學研究幽門螺旋桿菌引發之細胞內蛋白變化 螺旋桿菌引發之細胞內蛋白變化 / Sub-cellular a/ Sub-cellular and functional proteomic studies of cellular proteind functional proteomic studies of cellular proteins in response to Helicobacter pylorins in response to Helicobacter pylori

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實驗室成員實驗室成員 博三：陸德齡博三：陸德齡 碩二：郭芳婷、黃文智碩二：郭芳婷、黃文智 碩一：曾凱莉、林秋妏碩一：曾凱莉、林秋妏 大四：歐世宸、丁秀文大四：歐世宸、丁秀文

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ScheduleSchedule

Make competent cell 7/2~7/5 Make competent cell 7/2~7/5 Transformation & Mini prep 7/8~7/10Transformation & Mini prep 7/8~7/10 SDS-PAGE & Coomassie Blue Staining SDS-PAGE & Coomassie Blue Staining

7/19~7/22 7/19~7/22

Restriction digestion 7/18~7/19Restriction digestion 7/18~7/19 Midi prep 7/22~8/8Midi prep 7/22~8/8 Restriction digestion II 8/7~8/8Restriction digestion II 8/7~8/8 At The Bench 7/2~7/25At The Bench 7/2~7/25 報報 paper 9/5paper 9/5

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PrinciplePrinciple & Result & Result

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Make competent cellMake competent cell

先將先將 plateplate 上的上的 E. coliE. coli 重新培養到活性最高重新培養到活性最高的時期。的時期。

再用再用 0.1M CaCl0.1M CaCl22 resuspend resuspend 細胞兩次，使細胞兩次，使細胞成為細胞成為 competent cellcompetent cell 。。

急速冷凍到急速冷凍到 -70℃-70℃ 冰箱中。冰箱中。

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TransformationTransformation

取出冰凍的取出冰凍的 competent cellcompetent cell ，在冰上解凍。，在冰上解凍。 加入適量的加入適量的 plasmid DNAplasmid DNA 。。Heat shock(42 60sec)℃Heat shock(42 60sec)℃ 後馬上放回冰上。後馬上放回冰上。 再移到再移到 37 incubator℃37 incubator℃ 中培養。中培養。 將菌液塗到含有將菌液塗到含有 antibiotic antibiotic 的的 LB plateLB plate 上。上。 培養在培養在 37 incubator overnight℃37 incubator overnight℃

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Mini prepMini prep

離心後的離心後的 E.coli pelletE.coli pellet 分別加入分別加入 solution1solution1 、、22 、、 33

再離心後，留下再離心後，留下 supernatantsupernatant 。。 用用 95 Ethanol﹪95 Ethanol﹪ 沈澱沈澱 DNADNA 。。 離心使離心使 DNADNA 形成形成 pelletpellet 後，用後，用 70 Ethan﹪70 Ethan﹪

olol 清洗。清洗。 將將 DNADNA 溶到含有溶到含有 RNase RNase 的的 TE bufferTE buffer 中。中。 跑跑 DNA gelDNA gel 來確認所取的來確認所取的 DNADNA 。。

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1 No.2 pBJ β2-AR mini prep1 No.2 pBJ β2-AR mini prep2 No.1 pBJ β2-AR mini prep2 No.1 pBJ β2-AR mini prep3 pBJ β2-AR standard3 pBJ β2-AR standard4 Marker:1 Kb DNA Ladder4 Marker:1 Kb DNA Ladder5~8 5~8 秋妏學姊的秋妏學姊的 samplesample

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SDS-PAGESDS-PAGE

將待測的將待測的 protein sample protein sample 煮到煮到 denaturedenature 。。 加入加入 dyedye 後，後， loadload 到適當濃度的到適當濃度的 polyacrylpolyacryl

amide gelamide gel 中。中。 通電開始跑。通電開始跑。 跑完後，用跑完後，用 coomassie bluecoomassie blue 染。染。 再用再用 coomassie bluecoomassie blue 的溶劑的溶劑 distaindistain 、用水、用水

清洗後即可紀錄。清洗後即可紀錄。

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1.Marker:HMW-SDS 1.Marker:HMW-SDS standardstandard2.tube252.tube253.tube263.tube264.tube274.tube275.tube285.tube286.tube356.tube357.tube367.tube368.tube378.tube37

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Restriction digestionRestriction digestion

配好適量的反應濃度配好適量的反應濃度 在在 37℃37℃ 下作用足夠的時間下作用足夠的時間 在在 65 20min℃65 20min℃ 下終止下終止 enzymeenzyme 的活性的活性Run DNA gel to check resultRun DNA gel to check result

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1.pBJ β2-AR standard1.pBJ β2-AR standard

by Xho1by Xho1

2.pBJ β2-AR No.12.pBJ β2-AR No.1

by Xho1by Xho1

3.pBJ β2-AR No.23.pBJ β2-AR No.2

by Xho1by Xho1

4.Marker:1Kb DNA ladder4.Marker:1Kb DNA ladder

5.pBJ β2-AR standard5.pBJ β2-AR standard

6.pBJ β2-AR No.16.pBJ β2-AR No.1

7.pBJ β2-AR No.27.pBJ β2-AR No.2

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Midi prepMidi prep

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A.1Kb DNA LadderA.1Kb DNA Ladder

B.Load supernatantB.Load supernatant

C.Flow throughC.Flow through

D.Wash1D.Wash1

E.Wash2E.Wash2

F.EluteF.Elute

G.midi prepG.midi prep

H.pBJ HA-PAR4 standardH.pBJ HA-PAR4 standard

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Restriction Digestion 2Restriction Digestion 2_result1_result1

1.pBJ β2-AR S1.pBJ β2-AR S

2.pBJ β2-AR mini12.pBJ β2-AR mini1

3.pBJ β2-AR mini23.pBJ β2-AR mini2

4.pBJ β2-AR S by Xho14.pBJ β2-AR S by Xho1

5.pBJ β2-AR mini15.pBJ β2-AR mini1

by Xho1by Xho1

6.pBJ β2-AR mini26.pBJ β2-AR mini2

by Xho1by Xho1

7.1Kb DNA Ladder7.1Kb DNA Ladder

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Restriction Digestion 2Restriction Digestion 2_result2_result2

1.pET28a S1.pET28a S2.pET28a mini2.pET28a mini3.pET28a S by Xho13.pET28a S by Xho14.pET28a mini by Xho14.pET28a mini by Xho15.pBJ P4-P1T mini5 5.pBJ P4-P1T mini5 6.pBJ P4-P1T mini66.pBJ P4-P1T mini6

7 .pBJ P4-P1T mini5 by Xho1

8 .pBJ P4-P1T mini6 by Xho1

9.1Kb DNA Ladder

10.pBJ HA-PAR4 S

11.pBJ HA-PAR4 mini1

12.pBJ HA-PAR4 mini2

13.pBJ HA-PAR4 S by Xho1

14.pBJ HA-PAR4 mini1 by Xho1

15.pBJ HA-PAR4 mini2 by Xho2

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ProtocolProtocol

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Make competent cellMake competent cell

1.Streak plate and incubate at 37 overnight.℃1.Streak plate and incubate at 37 overnight.℃2.Pick single colony into the test tube with LB,incubate over2.Pick single colony into the test tube with LB,incubate over

night.night.3.Dilute the culture 1/100 into 50ml LB in 250 flask.Incubate 3.Dilute the culture 1/100 into 50ml LB in 250 flask.Incubate

until ODuntil OD600 600 reach 0.4~0.6.reach 0.4~0.6.

4.Centrifugation in 2000G 4 for 5min.℃4.Centrifugation in 2000G 4 for 5min.℃5.Resuspend the pellet with 12.5ml ice-cold 0.1M CaCl5.Resuspend the pellet with 12.5ml ice-cold 0.1M CaCl22..

6.Centrifugation in 2000G 4 for 5min.℃6.Centrifugation in 2000G 4 for 5min.℃7.Resuspend the pellet with 2ml CaCl7.Resuspend the pellet with 2ml CaCl22..

8.Fast-Freeze the cell in 158.Fast-Freeze the cell in 15 ％ ％ glycerol at –70 . ℃glycerol at –70 . ℃

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TransformationTransformation

1.Add 50ng plasmid DNA per 100μl competent cell.1.Add 50ng plasmid DNA per 100μl competent cell.Put them on ice for 30min.Put them on ice for 30min.

2. Water bath 42 60 sec.Put on ice 2 min.℃2. Water bath 42 60 sec.Put on ice 2 min.℃3.Remove to test tube containing 900λLB.Incubate 3.Remove to test tube containing 900λLB.Incubate

at 37 for 45 min.℃at 37 for 45 min.℃4.Smear proper volume of E. coli to LB agar plate 4.Smear proper volume of E. coli to LB agar plate

with antibiotic.with antibiotic.

5.Incubate at 37 overnight.℃5.Incubate at 37 overnight.℃

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Mini prepMini prep_1_1

1.Make the mini culture of target cell in the LB containing a1.Make the mini culture of target cell in the LB containing antibiotic.ntibiotic.

2.Gain the dry pellet by 12000G 4 30 sec.℃2.Gain the dry pellet by 12000G 4 30 sec.℃3.Resuspend the pellet with 100λSolution 1.3.Resuspend the pellet with 100λSolution 1.4.Add 200λSolution 2.Mix gently.4.Add 200λSolution 2.Mix gently.5.Add 150λSolution 3.Mix gently.5.Add 150λSolution 3.Mix gently.6.Put on ice for 5min.6.Put on ice for 5min.7.Centrifugation 12000G 4 15min.Remove the supernata℃7.Centrifugation 12000G 4 15min.Remove the supernata℃

nt to a fresh tube.nt to a fresh tube.8.Precipitate the DNA with 95 Ethanol.Put at R.T. for 2min.﹪8.Precipitate the DNA with 95 Ethanol.Put at R.T. for 2min.﹪

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Mini prepMini prep_2_2

9.Centrifugation 12000G 5min 4 .Decant the sup℃9.Centrifugation 12000G 5min 4 .Decant the sup℃ernatant.ernatant.

10.Rinse with 1ml 70 ethanol at 4 .Dry in the air.﹪ ℃10.Rinse with 1ml 70 ethanol at 4 .Dry in the air.﹪ ℃11.Dissolve the DNA with 20λTE buffer containing 11.Dissolve the DNA with 20λTE buffer containing

RNAse(2λRNAse/1ml TE).RNAse(2λRNAse/1ml TE).

12.Check the product by DNA agarose gel electrop12.Check the product by DNA agarose gel electrophoresis. horesis.

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SDS-PAGE & coomassie Blue StainingSDS-PAGE & coomassie Blue Staining_1_1

1.Preparation running gel assemblage.1.Preparation running gel assemblage.2.Add 12 separating gel,then add some ddwater t﹪2.Add 12 separating gel,then add some ddwater t﹪

o cover the gel,wait gel aggregated about 20min.o cover the gel,wait gel aggregated about 20min.3.Remove water phase.Add 5 stacking gel,put the 3.Remove water phase.Add 5 stacking gel,put the

slab into stacking gel, wait gel aggregated about slab into stacking gel, wait gel aggregated about 30 min.30 min.

4.Denature sample protein with boiling 100 wate℃4.Denature sample protein with boiling 100 wate℃r 10min.Then load sample to wells.r 10min.Then load sample to wells.

5.Running gel with constant 150V,maximum curre5.Running gel with constant 150V,maximum current(about 1.5 hr).nt(about 1.5 hr).

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SDS-PAGE & coomassie Blue StainingSDS-PAGE & coomassie Blue Staining_2_2

6.Take gel,remove stacking gel,then put into Coom6.Take gel,remove stacking gel,then put into Coomassie Blue Staining solution for 10min.assie Blue Staining solution for 10min.

7.Destain with DS-1 for 20min.7.Destain with DS-1 for 20min.

8.Destain with DS-2 for 30min(until background is 8.Destain with DS-2 for 30min(until background is clear).clear).

9.Wash with ddwater 2 times.9.Wash with ddwater 2 times.

10.Record the gel image and conserve the gel.10.Record the gel image and conserve the gel.

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Midi prepMidi prep_1_1

1.Streak plate,pick single colony to make mini culture.1.Streak plate,pick single colony to make mini culture.2.Dilute 1/1000 to a flask with 2.Dilute 1/1000 to a flask with 50ml50ml LB and antibiotic. LB and antibiotic.3.Centrifugation 3000G 30min 4 to get the pellet.℃3.Centrifugation 3000G 30min 4 to get the pellet.℃4.Resuspend with Buffer P1.4.Resuspend with Buffer P1.5.Add Buffer P2,then incubate at R.T. 5.Add Buffer P2,then incubate at R.T. 6.Add Buffer P3,incubate on ice.6.Add Buffer P3,incubate on ice.7.Centrifugation 20000G 4 ,remove the supernatant ℃7.Centrifugation 20000G 4 ,remove the supernatant ℃

to another centrifuge tube.to another centrifuge tube.8.Re-centrifuge,remove the supernatant.*8.Re-centrifuge,remove the supernatant.*9.Let Buffer QBT flow through the QIAGEN-tip 100 9.Let Buffer QBT flow through the QIAGEN-tip 100

column.column.

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Midi prepMidi prep_2_2

10.Take the supernatant of step 8 to flow through column.*10.Take the supernatant of step 8 to flow through column.*

11.Use Buffer QC to wash column two times.*11.Use Buffer QC to wash column two times.*

12.Elute DNA with Buffer QF.12.Elute DNA with Buffer QF.

13.Precipitate DNA by adding isopropanol at R.T.Centrifugation 13.Precipitate DNA by adding isopropanol at R.T.Centrifugation and wash with 70 ethanol.﹪and wash with 70 ethanol.﹪

14.Centrifugation , air dry the pellet ,and redissolve DNA in prop14.Centrifugation , air dry the pellet ,and redissolve DNA in proper volume of TE buffer.er volume of TE buffer.

15.Test the concentration of product , calculate the yield,and run 15.Test the concentration of product , calculate the yield,and run DNA gel to check the purification.DNA gel to check the purification.

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Restriction DigestionRestriction Digestion

1.1. 設計好反應溶液的量：包括設計好反應溶液的量：包括 11 倍的倍的 NEB 2NEB 2 、、 11 倍的倍的 BSABSA 、、適量的適量的 DNADNA 及足夠的及足夠的 enzymeenzyme ，於，於 total 10λtotal 10λ 的溶液中。的溶液中。

2.Water bath at 37 for more than 2hr.℃2.Water bath at 37 for more than 2hr.℃3.Water bath at 65 for 20 min.℃3.Water bath at 65 for 20 min.℃4.Run DNA gel 4.Run DNA gel