Editing Colony Screening

1. Take 10&nbsp;µl ddH<sub>2</sub>O into each 1.5 ml tube. 

2. Pick colonies from plate with pipette tips, dip the tips into the tube with 10&nbsp;µl ddH<sub>2</sub>O,&nbsp; pipette up and down several times;&nbsp;&nbsp; then take 3&nbsp;µl into a new PCR tube, 

&nbsp;&nbsp;&nbsp; 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.&nbsp; 

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

&nbsp;&nbsp;&nbsp; 10X PCR buffer, with Mg<sup>2+</sup>: 2.0 µl * N<br> 

&nbsp;&nbsp;&nbsp; 10mM dNTP: 0.5 µl * N<br>&nbsp;&nbsp;&nbsp; Vector specific forward primer: 0.5 µl * N<br>&nbsp;&nbsp;&nbsp; Insert (gene) specific reverse primer: 0.5 µl * N<br>

&nbsp;&nbsp;&nbsp; (or Vector specific reverse primer + insert (gene) specific forward primer)<br>

&nbsp;&nbsp; Taq enzyme (do not use Pfu or Phusion): 0.5 or 1 unit * N<br>&nbsp;&nbsp; ddH2O: 13.5 µl&nbsp; *&nbsp; N 

&nbsp;&nbsp;&nbsp; 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 <sup>o</sup>C.<br>

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

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 <sup>o</sup>C or 37 <sup>o</sup>C) overnight.<br> 

[[Category:Protocols]]