Crm at 20:41, 9 September 2011

2011-09-09T20:41:34Z

New page

1. Take 10 µl ddH2O into each 1.5 ml tube.

2. Pick colonies from plate with pipette tips, dip the tips into the tube with 10 µl ddH2O,  pipette up and down several times;   then take 3 µl into a new PCR tube,

    After finishing PCR assembly, add 500 µl of LB/Kan or LB/Amp to 7 µl bacterial suspension, put on a eppendorf mixer, shake at 37 oC for several hours. 

3. First prepare PCR master mix (for N samples)

    10X PCR buffer, with Mg2+: 2.0 µl * N 

    10mM dNTP: 0.5 µl * N     Vector specific forward primer: 0.5 µl * N     Insert (gene) specific reverse primer: 0.5 µl * N 

    (or Vector specific reverse primer + insert (gene) specific forward primer) 

   Taq enzyme (do not use Pfu or Phusion): 0.5 or 1 unit * N    ddH2O: 13.5 µl  *  N

    Total 20 µl * N

4. Take 17 µl master mix to each PCR tube.

5. PCR, make sure use 1 min per kb extensionat 72 oC. 

6. Add 4 µl of 6X Loading Dye, Run gel 

7. Transfer 300 µl bacteria suspension to 15 ml falcon tube, add 4 ml LB/Kan or LB/Amp, grow bacteria in a shaker incubator (30 oC or 37 oC) overnight. 

[[Category:Protocols]]

Colony Screening - Revision history

Crm at 20:41, 9 September 2011