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.


PCR

1. Dissolve the primers in water to have the concentration of 10nM.


2. PCR reaction mixer： 

 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


3. Put the reaction mixer in a PCR tube.

4.The PCR program is as follow :

4.1
94℃ 30 seconds
60℃ 30seconds
72℃ 2 minutes
Cycle 9 times


4.2
94℃ 30 seconds
55℃ 30seconds
72℃ 2 minutes
Cycle 34 times

Extend PCR product at 72℃ for 10 minutes


5. The PCR product was examed by electrophoresis in 1％ agarose.



T-A cloning protocol

Take an eppendorf tube and add following component one by one and mix well,incubated at 4℃ overnight. 


 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(To verify the presence of our gene of interest)

  1. Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.
  2. Discard the supernatant
  3. Add 500μl ddH20, Vortex
  4. Boil for 20 min.
  5. Take 5μl of above bacterial lysate, and add 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 product was examed by electrophoresis in 1％ agarose.

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