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Inverse PCR

Inverse PCR

For use with Snyder mTn-lacZ/LEU2 based mutagenesis

  1. Genomic DNA Prep
    • from 5 ml culture, resuspend in 50 l TE

  2. Digestions
    • Genomic DNA5 l
      10x NEB Buffer5 l
      0.5 g/l RNaseA1 l
      H2O38 l
      Restriction Enzyme1 l
      • Use either AciI, AluI, HaeIII, HpaII, RsaI or TaqI for Leu transposon libraries
      • 37 deg C/ >3 hours (or overnight)
      • 65 deg C/ 20 min

  3. Ligations (intramolecular, hopefully)
    • Digested DNA10 l
      10x Ligation Buffer20 l
      H2O~170 l
      T4 DNA Ligase (NEB, 400U/l)0.2 l
      • all day at room temp or overnight at 4 deg C
      • precipitate with 80 l 5M NH4Ac + ethanol…-20 deg C/ >1 hr; spin, dry, resuspend pellet in 100 l TE

  4. PCR
    • ligated DNA10 l
      3 M KCl0.75 l
      1 M Tris, pH 8.50.4 l
      25 mM MgCl23 l
      10 mM dNTPs1 l
      10 M oligo#1*1 l
      10 M oligo#2*1 l
      H2O32.35 l
      Taq (added after hot start)1 l
      • 35 cycles of: 94 deg C/1', 62 deg C/1', 72 deg C/2'30"
      • *the choice of oligos used for the PCR reaction depends on what restriction enzyme was initially chosen to cut the genomic DNA and what "side" of the transposon you want to use as the starting point for the PCR.

      EnzymeOligos for PCR
      AciIInPCR3 and InPCR4
      AluIInPCR3 and InPCR4
      HaeIIIInPCR3 and InPCR4
      HpaIIInPCR3 and InPCR4
      RsaIInPCR1 and InPCR2 or
      InPCR4 and InPCR5
      TaqIInPCR1 and InPCR2 or
      InPCR4 and InPCR6

        InPCR1 => 5'-taagttgggtaacgccagggttttc-3'
        InPCR2 => 5'-ttccatgttgccactcgctttaatg-3'
        InPCR3 => 5'-ataactacgatacgggagggcttacc-3'
        InPCR4 => 5'-gattaagcattggtaactgtcagacc-3'
        InPCR5 => 5'-cataattctcttactgtcatgccatcc-3'
        InPCR6 => 5'-tcaaggatcttaccgctgttgagatcc-3'

  5. Cleanup for Sequencing (2 options)
    1. Wizard PCR purification spin columns
      • elute in 50 ul TE
    2. Exonuclease I+ shrimp alkaline phosphatase treatment:
      • In PCR tubes, add:
      • 8.5 l PCR product
      • 1 l Exonuclease I
      • 1 l SAP (shrimp alkaline phosphatase)
        • 37 deg C/20 min
          65 deg C/20 min

  6. Sequencing
    • Cleaned DNA10.5 l
      Sequencing Mix8 l
      DMSO1 l
      Sequencing Oligo, 10 M0.5 l
      • use Amersham cycle seq protocol (96 deg C/30", 45 deg C/15", 60 deg C/4'; 30 cycles for dilute templates)
      • after cycling, put rxn through a Pharmacia Auto-Seq G-50 column, and dry
      • if using the Exo/SAP treatment above, then just use the entire reaction for sequencing
      • for sequencing from InPCR1-2 products, use mTn3-SEQ1 oligo
        mTn3-SEQ1 => 5'-cccccttaacgtgagttttcgttccact-3'
      • for sequencing from InPCR3-4, InPCR4-5, or InPCR4-6 products use mTn3-SEQ2 oligo
        mTn3-SEQ2 => 5'-aaggatctaggtgaagatcc-3'
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