Inverse PCR

For use with Snyder mTn-lacZ/LEU2 based mutagenesis

  1. Isolate genomic DNA as in the Yeast Genomic Prep protocol.

  2. Digest genomic DNA as follows:

    • 38 ul distilled water
    • 5 ul genomic DNA
    • 5 ul appropriate 10x NEB buffer
    • 1 ul 0.5 mg/ml RNAse
    • 1 ul desired restriction enzyme (Use either AciI, AluI, HaeIII, HpaII, RsaI or TaqI for Leu transposon libraries).

    1. Incubate digests at 37oC (or appropriate temperature) for 3-18 hours.
    2. Incubate digests at 65oC to inactivate the restriction enzymes.
  1. Ligate digests as follows:

    • 170 ul distilled water
    • 10 ul digested genomic DNA
    • 20 ul 10x NEB ligation buffer
    • 1 ul T4 DNA ligase

    1. Incubate ligations at room temperature 6-18 hours.
    2. Add 80 ul 5M NH4OAc and 700 ul ethanol. Incubate 1-3 hours at -20oC.
    3. Pellet DNA by microcentrifugation at 14,000 rpm for 10 minutes. Aspirate off supernatant.
    4. Pulse-spin to collect remaining supernatnt. Aspirate supernatant from DNA.
    5. Resuspend in 100 ul TE.
  1. PCR as follows:

    • 32.35 ul distilled H2O
    • 0.75 ul 3M KCl
    • 0.4 ul 1M Tris pH8.5
    • 3 ul 25mM MgCl2
    • 1 ul 10 mM dNTPs
    • 1 ul 10uM oligo1
    • 1 ul 10uM oligo2
    • 10 ul ligated DNA
    • 1 ul Taq Polymerase

      Note: 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.

      Enzyme

      Oligos for PCR

      AciI

      InPCR3 and InPCR4

      AluI

      InPCR3 and InPCR4

      HaeIII

      InPCR3 and InPCR4

      HpaII

      InPCR3 and InPCR4

      RsaI

      InPCR1 and InPCR2 or
      InPCR4 and InPCR5

      TaqI

      InPCR1 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'
      Cycles:

      95oC for 5 minutes

      95oC for 1 minute
      62oC for 1 minute
      72oC for 3 minutes
        ==> 35 cycles

      72oC for 10 minutes
      4oC hold
  1. Clean-up DNA for sequencing as follows:

    1. Purify DNA using Wizard PCR purification spin columns (Promega).
    2. Elute DNA in 50 ul TE.
    3. To 8.5 ul cleaned DNA, add 1 ul Exonuclease I and 1 ul Shrimp Alkaline Phsphatase.
    4. Incubate at 37oC for 20 minutes.
    5. Heat inactivate enzymes by incubation at 65oC for 20 minutes.
  1. Sequencing as follows:

    • 8 ul Sequencing Mix
    • 1 ul DMSO
    • 10.5 ul cleaned DNA
    • 0.5 ul 10 uM sequncing oligo.

      NOTE: 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 => 5'-cccccttaacgtgagttttcgttccact-3'

      For sequencing from InPCR3-4, InPCR4-5, or InPCR4-6 products use mTn3-SEQ2 oligo => 5'-aaggatctaggtgaagatcc-3'

      Cycles: 95oC for 5 minutes

      96oC for 30 seconds
      45oC for 15 seconds
      60oC for 4 minutes
        ==> 30 cycles

      4oC hold