Supplemental Protocol 2: Using colony PCR to identify positive clones (NEB #M0689)

This protocol provides a quick way to examine how correct cloned sequences are without additional time waiting for cell growth and plasmid minipreps. Amplicons can be obtained in a few hours from single colony PCR and cleaned-up, followed by DNA sequencing.

1. Identify colonies.
   
   
   1. Identify colonies containing full length of gene of interest without overnight culturing, purification of plasmid DNA, and restriction digest or Sanger sequencing. Pick up 6 colonies from the outgrowth plate, replica plate onto appropriate selective plate, and transfer remaining colony into individual PCR tubes containing the following components:
      
      

      REAGENT	REACTION	FINAL RXN CONC.
      OneTaq Hot Start Quick-Load 2X Master Mix	12.5 μl	1X
      10 μM Forward Primer	0.5 μl	0.2 µM
      10 μM Reverse Primer	0.5 μl	0.2 µM
      Nuclease-free water	11.5 μl
      Colony	1 colony

      
      
      
2. Setup PCR reaction conditions:
   
   
   1. PCR reaction conditions:
      
      

      CYCLE STEP	TEMP	TIME	CYCLES
      Initial Denaturation	95°C	5 minutes
      Denaturation	95°C	15 seconds	33
      Annealing	55–64°C*	15 seconds
      Extension (for 500–1,000 bp)	72°C	30–60 seconds
      Final Extension	72°C	5 minutes
      Hold	4°C

      * Please visit tmcalculator.neb.com to determine correct annealing temperature.
      
      

3. Identify positive clones.
   
   
   1. Examine the size of PCR amplicons (5–10 µl) on an agarose Select 4 amplicons with the anticipated size of gene of interest to proceed.
      
      
   2. Clean up these reactions using Exo-CIP Rapid PCR Cleanup Kit (NEB #E1050) or go back to the replica plate (after overnight incubation) to start cultures for miniprep DNA preparation (e.g., Monarch PCR & DNA Cleanup Kit (5 µg – NEB #T1030)).
      
      
   3. Submit Exo-CIP treated PCR reactions or plasmid DNA for Sanger sequencing with appropriate primers.