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Colony PCR protocol

About the Figure: Fast colony PCR helps identify fungal mutants. While studying the molecular mechanism of gene targeting in the mushroom Coprinopsis cinerea (shown in color in the image above), researchers used SapphireAmp® Fast PCR Master Mix to identify gene disruption mutants by colony PCR. Nakazawa, T., et al. (2011) Fungal Genetics Biol. 48:939.

Streamline your colony PCR protocol to mere minutes

Colony PCR is a method used to screen for plasmids containing a desired insert directly from bacterial colonies without the need for culturing or plasmid purification steps. SapphireAmp Fast PCR Master Mix further improves the colony PCR workflow by allowing a streamlined protocol:

• Assemble PCR reactions by simply adding primers and cells to the master mix
• After PCR, load directly onto a gel for electrophoresis
• Compatible with direct restriction enzyme digestion of PCR products
• Screen inserts of up to 2 kb in only 60 minutes
  Try a sample of SapphireAmp Fast PCR Master Mix

Experimental Example: Using SapphireAmp Fast PCR Master Mix to Screen a cDNA Library by Colony PCR

colony PCR protocol data
Figure 1. Colony PCR with SapphireAmp Fast PCR Master Mix.
  E. coli cells were transformed with a mouse cDNA library (insert size from 0.5 to 5 kb) ligated into pUC118. Plasmid-containing transformants were analyzed using SapphireAmp Fast PCR Master Mix and M4 and RV primers. The PCR reaction cycle was complete in just 70 minutes, and excellent yields were obtained. All 16 clones checked were confirmed to contain 0.5 to 5 kb inserts. For additional experimental details, click here to download the entire application note.

Overview of Colony PCR Protocol

colony PCR protocol overview Figure 2. Colony PCR protocol with SapphireAmp Fast PCR Master Mix. Colony PCR can be set-up with three basic steps: 1. pick a colony, 2. "poke" to a replica plate, and 3. perform fast PCR using suspended E. coli cells as template.  

Speed up Colony PCR with SapphireAmp Fast PCR Master Mix

SapphireAmp Fast PCR Master Mix is fast— with an extension speed of 10 seconds/kb, reactions can be completed in half the time of conventional Taq. SapphireAmp master mix is also significantly faster than dye-added premixes offered by other manufacturers. The table below lists the reaction time for SapphireAmp master mix and the recommended reaction times for two other commercially available dye-added premixes.

Amplicon Length SapphireAmp Fast PCR
Master Mix
Company P dye-added master mix Company T dye-added master mix
0.5 kb 50 min 1 hr 35 min 1 hr 30 min
1.0 kb 55 min 1 hr 50 min 1 hr 45 min
2.0 kb 1 hr 2 hr 20 min 2 hr 15 min
4.0 kb 1 hr 30 min 3 hr 20 min 3 hr 15 min


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