Colony Screening

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.