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SapphireAmp Fast PCR Master Mix SapphireAmp Fast PCR Master Mix
Tech Note

Colony PCR: Protocol complete in under an hour

Streamline your colony PCR protocol to mere minutes

  • Simple PCR assembly: Just add primers and cells to the master mix
  • Save time: After PCR, load reactions directly onto a gel
  • Restriction enzyme-friendly: Digest PCR products directly—no need for gel purification or buffer exchange
  • Exceptionally fast: Screen inserts of up to 2 kb in only 60 minutes
  • Accelerate your cloning: Use in conjunction with In-Fusion Cloning systems to pair fast, accurate cloning with streamlined screening
Overview Results Methods

Overview  

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. With SapphireAmp Fast PCR Master Mix, the colony PCR workflow is further improved and 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 the template
Colony PCR protocol overview

Figure 1. Colony PCR protocol with SapphireAmp Fast PCR Master Mix. 

Results  

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 reaction times for SapphireAmp master mix and the recommended reaction times for two other commercially available dye-added premixes.

Amplicon lengthSapphireAmp Fast PCR Master MixCompany P dye-added master mixCompany 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


Experimental example: Using SapphireAmp Fast PCR Master Mix to screen a cDNA library by colony PCR

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 (see Methods below). 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.

Colony PCR protocol data

Figure 2. Colony PCR with SapphireAmp Fast PCR Master Mix.

Methods  

PCR master mix was prepared by including the following components (per reaction): SapphireAmp Fast PCR Master Mix (2X Premix), 25 µl; M13 Primer M4 (20 µM), 0.5 µl; M13 Primer RV (20 µM), 0.5 µl; dH2O, 24 µl. PCR master mix aliquots (50 µl) were dispensed into each PCR tube. A sterile micropipette tip was used to transfer a few cells from each colony to a corresponding PCR tube, where cells were resuspended in PCR master mix. PCR was performed using a Takara PCR Thermal Cycler Dice thermal cycler (not available in all geographic locations). The cycling conditions were: 94°C, 1 min; followed by 30 cycles of 98°C, 5 sec; 55°C, 5 sec; and 72°C, 40 sec. After PCR was complete, 5 µl of each reaction was subjected to electrophoresis on a 1% L03 agarose gel.

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