See Colony PCR for general information about this protocol and other variants
- Sterile 0.6mL plastic tubes
- LB-agar plate with appropriate antibiotic
- Oligonucleotide pair (usually VF2 and VR)
- PCR supermix
- PCR machine
- Sterile pipet tips
- Prepare one sterile 0.6mL tube with 20μL ddH2O for each colony you intend to pick.
- Prepare LB-agar plate with appropriate antibiotic to use as index plate.
- Pick single colony using a pipetman with sterile tip. The pipettor should be set to 3μL
- Inoculate tip with colony into tube. Pipet up and down to ensure cells are transferred to tube. Pipet 3μL of cells suspended in water onto index plate.
- Repeat for as many colonies as you intend to pick.
- 9 μL PCR supermix
- 0.25 μL 40nM VF2
- 0.25 μL 40nM VR
- 0.5 μL colony template
- 95°C for 15 mins
- 94°C for 30 secs
- 56°C for 30 secs
- 68°C for 1 min per kb of expected product
- Repeat 2-4 39 times.
- 68°C for 20 mins
- 4°C forever
Run a gel to determine amplification product length.
- Lately, I've been carrying out this procedure using a multichannel pipettor to speed steps up. I use a P50 multichannel pipettor to add 20μL water to the appropriate number of wells in a 96 well plate/ Picking colonies is essentially the same except that I inoculate the colony into a 96 well plate rather than a tube. The PCR is carried out in 8 tube strips rather than individual tubes. After adding 9.5μL of the primer + PCR supermix master mix to each PCR tube, I transfer 1μL of colony-water mixture to the reaction tube using a P10 multichannel pipettor. (I transfer 1 μL rather than 0.5μL because it is easier to pipette.) I then touch spin the PCR tube strips in a rack using a swinging bucket rotor in order to collect the contents of the tube to the bottom. The PCR conditions are the same. -- RS