CRI colony PCR

General Info

This SOP is a how to guide for PCR from a single colony selected by hand using a toothpick.

Materials

• Toothpicks
• Orbital Incubator Shakers
• PCR tubes
• PCR reagents
• MJ Research PCR machines

Procedure

1. 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.
   1. Remove 2.5µl and return to incubate o/n for Plasmid/Cosmid DNA minipreps of positives for sequencing
   2. Revise antibiotic for specific vector
2. Prepare PCR Mastermix
   1. 25µl total reaction volume
   2. 1.25µl DMSO
   3. 2.5µl WhiB-F primer (1.5µM 10ng/µl)
   4. 2.5µl WhiB-R primer (1.5µM 10ng/µl)
   5. 12.5µl Qiagen Hotstart Mastermix
   6. 6.25µl dH20
   7. Adjust for number of samples required + one for volume/carry over error.
   8. Include positive control DNA sample and negative control no DNA sample.
3. Dispense 22.5µl of Mastermix into PCR tubes
4. Add 2.5µl of each bacterial culture into PCR tubes
5. Run the PCR reactions using the following programme:
6. PCR Programme: Beth-Colony
   1. 15 min 95ºC x 1 cycle
   2. 30 sec 94ºC |
   3. 45 sec 53ºC | x 29 cycles
   4. 30 sec 72ºC |
   5. 10 min 72ºC x 1 cycle
   6. ~ 4ºC
   7. Adjust annealing temperature to the optimum for the specific primers
   8. Adjust extension time to the optimum for the product size (30 sec/500bp)
7. Run 5µl of PCR product on an E-gel (JGL-SOP-035 and JGL-SOP-036)for 15 min.