Team:TzuChiU Formosa/Protocol

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Revision as of 13:04, 20 October 2009

Protocol

Tansformation Protocol

1. Take the cp919 competent cell eppendorf from -80℃ freezer.
2. Add 4ul plasmid to competent cell and place at ice for 30 minutes.
3. Put the eppendorf into 42℃ water bath for 90 seconds.
4. Place the eppendorf at ice for 2 minutes.
5. Add 1ml LBM to the eppendorf and well mixed.
6. Put the eppendorf into 37℃ water bath incubate for an hour.
7. Centrifuged at 7000 r.p.m for 5 minutes and remove some supernatant liquid.
8. Spread antibiotic which we need on plate. When the plate dry, spread appropriate the culture liquid on plate.
9. Incubate into 37℃ water bath for 16~18 hours.

Competent Cell (CP919-Cph8)

1. Day1. Streak out the E.coli strain on an LBM plate (with kanamycin antibiotic) to isolate colonies and incubate at 37℃ overnight (16-20 hours).
2. Day2. Select a single colony and inoculate 10 ml sterile LBM Grow overnight (16-20 hours) in a 37℃ shaker incubator.
3. Day3. Add 2ml overnight culture to 250ml flask which with 100 ml sterile LBM.
4. Grow the cultures to OD600 = 0.2~0.4 (incubate about 75~90 min.).
5. Centrifuge, 4℃,3000 r.p.m,10 min.
6. Discard the supernatant and mix the cell pellet with 10ml FSB.
7. Keep the cells on ice for 3~4 hours.
8. Centrifuge, 4℃,3000 r.p.m,10 min.
9. Discard the supernatant and mix the cell pellet with 5ml FSB.
10. Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf. Freeze these tubes on N2 (aq.) and then transfer them to -80℃ freezer.

Real Time PCR

1. Take ice for preparing.

2. dilution of concentration：

2.1

template DNA（Omp R）：500 times diluted

499μl ddH2O ＋ 1μl omp R＝500μl

2.2

primer forward ＆ primer reverse（p88）：10 times diluted

10μl p88 primer forward＋ 90μl ddH2O

3. Materials：

 Template DNA（10ng/μl）		5
 10× PCR buffer			2
 10× dNTP（2mM）			2
 forward primer（10μM）		0.5
 reverse primer（10μM）		0.5
 Pfu DNA polymerase（2Kb）	0.1
 PCR water			9.9
 _______________________________________
 Total				20 μl

4. Step：

Meacure template DNA 2μl Measure 10× PCR buffer 2μl Measure 10× dNTP 2μl Measure forward primer 0.5μl Measure reverse primer 0.5μl Measure Pfu DNA polymerase 0.1μl Follow this step ，puttng the reagent in the eppendorf.

5. Use the PCR to amplify our product：PCR program

5.1

94℃ 30 seconds

60℃ 30seconds

72℃ 2 minutes

Cycle 9 times

5.2

94℃ 30 seconds

55℃ 30seconds

72℃ 2 minutes

Cycle 34 times

72℃ 10 minutes

6. The PCR production separate by 1％ agar.

7. After separate using the EtBr to dye agar and exposing by UV.

T-A cloning protocol

Take an eppendorf,and add these one by one,after mix well,overnight 4℃.

 2x ligase buffer	7.5μl
 Insert(Aeq.-GFP)	5.5μl
 Vector(pGEM-T-easy)	1μl
 T4 DNA exp 3/12	1μl
 _________________________________
 total			15μl

Cloning PCR

1. Add 10μl cell, centrifuge 14000 rpm, 10 min
2. Discard the supernatant
3. Add 500μl ddH20, Votex
4. Boiling 20min, 100℃
5. Add 5μl DNA, 10x dNTP 2μl, 10x Buffer 2μl, forward primer（10μM）0.5μl reverse primer（10μM）0.5μl, Taq DNA polymerase 0.1μl, ddH20
6. Use the PCR to amplify our product：PCR program

 95℃ 4 min 
 94℃ 30seconds 
 55℃ 40seconds 
 72℃ 2 min
 Cycle 34 times
 25℃ 2 min

　7. The PCR production separate by 1％ agar.

　8. After separate using the EtBr to dye agar and exposing by UV.

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