Colony PCR: Protocol Complete in Under an Hour
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:
- 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 of Colony PCR Protocol
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. Download a short protocol.
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|
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 with SapphireAmp Fast PCR Master Mix.
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.