Insert Screening Protocols for NEB PCR Cloning Kit
Many DNA polymerases are suitable for colony PCR. For inserts < 1 kb, NEB recommends OneTaq DNA Polymerase (NEB #M0480) or OneTaq Hot Start DNA Polymerase (NEB #M0481). Taq DNA Polymerase (NEB #M0267) can also be used. For inserts > 1 kb NEB recommends OneTaq formulations, LongAmp Taq DNA Polymerase (NEB #M0323) or LongAmp Hot Start Taq DNA Polymerase (NEB #M0534); the LongAmp Taq formulations are especially useful for larger inserts. These products are also available in easy-to-use master mix formats. Following your choice of DNA polymerase, use the provided sequence of the cloning analysis forward and reverse primers and the Tm calculator found on the NEB website (TMCalculator.neb.com) to determine the annealing temperature for your PCR reactions.
Screening Protocol 1: Colony PCR using OneTaq 2X Master Mix with Standard Buffer (NEB #M0482) or LongAmp Taq 2X Master Mix (NEB #M0287)
- Prepare a PCR mix of sufficient volume to allow 50 μl per screened colony:
AMOUNT FINAL CONC. OneTaq or LongAmp Taq 2X Master Mix 25.0 μl 1 X Cloning Analysis Forward Primer (100 μM) 0.15 μl 300 nM Cloning Analysis Reverse Primer (100 μM) 0.15 μl 300 nM H2O 24.7 μl Total to 50 μl
- Use a sterile toothpick or pipette tip to pick an individual colony, and dip into each amplification reaction. To create a stock of each individual colony, either dip the same toothpick or pipette tip into 3 ml growth media containing ampicillin, or use a separate agarose-ampicillin plate to prepare a streak or patch of the colony material.
2a. Transfer the reactions to a thermocycler and perform PCR with the following conditions:
STEP TEMP TIME Initial Denaturation 94°C 2 minutes 30 Cycles 94°C 15 seconds 53–57°C* 15 seconds 68°C 60 seconds/kb Final Extension 68°C 5 minutes Hold 4–10°C ∞
- Load 5 μl of each completed PCR onto an agarose gel alongside an appropriate DNA ladder [e.g., Quick-Load Purple 1 kb Plus DNA Ladder (NEB #N0550)]. For reference, the amplicon length in the absence of an insertion would be 309 bp in length. The amplicon length with the positive control insert would be 1312 bp.