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.
 * 2) Remove 2.5µl and return to incubate o/n for Plasmid/Cosmid DNA minipreps of positives for sequencing
 * 3) Revise antibiotic for specific vector
 * 4) Prepare PCR Mastermix
 * 5) 25µl total reaction volume
 * 6) 1.25µl DMSO
 * 7) 2.5µl WhiB-F primer (1.5µM 10ng/µl)
 * 8) 2.5µl WhiB-R primer (1.5µM 10ng/µl)
 * 9) 12.5µl Qiagen Hotstart Mastermix
 * 10) 6.25µl dH20
 * 11) Adjust for number of samples required + one for volume/carry over error.
 * 12) Include positive control DNA sample and negative control no DNA sample.
 * 13) Dispense 22.5µl of Mastermix into PCR tubes
 * 14) Add 2.5µl of each bacterial culture into PCR tubes
 * 15) Run the PCR reactions using the following programme:
 * 16) PCR Programme: Beth-Colony
 * 17) 15 min 95ºC x 1 cycle
 * 18) 30 sec 94ºC |
 * 19) 45 sec 53ºC |  x 29 cycles
 * 20) 30 sec 72ºC |
 * 21) 10 min 72ºC x 1 cycle
 * 22) ~ 4ºC
 * 23) Adjust annealing temperature to the optimum for the specific primers
 * 24) Adjust extension time to the optimum for the product size (30 sec/500bp)
 * 25) Run 5µl of PCR product on an E-gel (JGL-SOP-035 and JGL-SOP-036)for 15 min.