Team:TzuChiU Formosa/Protocol

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Contents

Protocol

Project.jpg

Tansformation Protocol

  1. Take the cp919 competent cell in an eppendorf tube from -80℃ freezer put in ice.
  2. Add 4ul plasmid to competent cell and place in ice for 30 minutes.
  3. Put the transformed cells into 42℃ water bath for 90 seconds.
  4. Then place the cells in ice for 2 minutes.
  5. Add 1ml LB to the cells and mixed.
  6. Put the eppendorf tube in 37℃ water bath and incubate for an hour.
  7. Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.
  8. While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.
  9. Incubate at 37℃ incubater for 16~18 hours.

Competent Cell (CP919-Cph8)

  1. Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).
  2. Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.
  3. Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.
  4. Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)
  5. Spin down the bacteria at 4℃,3000 rpm for 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. Spin down, at 4℃,3000 rpm for 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 tubes. Freeze these tubes in liquid nitrogen, 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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