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  • Insert Screening Protocols for NEB PCR Cloning Kit

    Screening for inserts can be performed by colony PCR, restriction digestion or PCR of mini-prep plasmid DNA, or by sequencing of the mini-prep plasmid DNA.

    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 or LongAmp Taq 2X Master Mix

    1. Prepare a PCR mix of sufficient volume to allow 50 μl per screened colony:
      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

    2. 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:
        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
        *For OneTaq, use 53°C; for LongAmp, use 57°C
    3. Load 5 μl of each completed PCR onto an agarose gel alongside an appropriate DNA ladder [e.g., Quick-Load Purple 2-Log DNA Ladder (NEB #N0550)]. For reference, the amplicon length in the absence of an insertion would be 246 bp in length.

    Screening Protocol 2: Sequence Analysis

    The Cloning Analysis Forward and Reverse Primers can also be used for sequencing inserts. This can be performed with purified plasmids from overnight cultures from each colony, or with amplicons from the above colony screening PCRs. The primers anneal 110 bp upsteam and 91 bp downsteam (measured from the 3´ end of each primer to cloning insertion site), ensuring complete reads of the insert.