CRI colony PCR
This SOP is a how to guide for PCR from a single colony selected by hand using a toothpick.
- Orbital Incubator Shakers
- PCR tubes
- PCR reagents
- MJ Research PCR machines
- Toothpick colonies/glycerol in 1.2 ml 2 x YT + 50µg/ml Kan for 2 h at 37 ºC shaking in orbital shaker (JGL-SOP-XXX). The medium should look only slightly turbid. Do not grow the cultures for longer because the accumulation of metabolites will interfere with the PCR reaction.
- Remove 2.5µl and return to incubate o/n for Plasmid/Cosmid DNA minipreps of positives for sequencing
- Revise antibiotic for specific vector
- Prepare PCR Mastermix
- 25µl total reaction volume
- 1.25µl DMSO
- 2.5µl WhiB-F primer (1.5µM 10ng/µl)
- 2.5µl WhiB-R primer (1.5µM 10ng/µl)
- 12.5µl Qiagen Hotstart Mastermix
- 6.25µl dH20
- Adjust for number of samples required + one for volume/carry over error.
- Include positive control DNA sample and negative control no DNA sample.
- Dispense 22.5µl of Mastermix into PCR tubes
- Add 2.5µl of each bacterial culture into PCR tubes
- Run the PCR reactions using the following programme:
- PCR Programme: Beth-Colony
- 15 min 95ºC x 1 cycle
- 30 sec 94ºC |
- 45 sec 53ºC | x 29 cycles
- 30 sec 72ºC |
- 10 min 72ºC x 1 cycle
- ~ 4ºC
- Adjust annealing temperature to the optimum for the specific primers
- Adjust extension time to the optimum for the product size (30 sec/500bp)
- Run 5µl of PCR product on an E-gel (JGL-SOP-035 and JGL-SOP-036)for 15 min.