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RESEARCH DIVISION Laboratory Manual

 


 

Inverse PCR (To Isolate DNA Adjacent To Known Sequence in Genomic DNA)

  1. Design primers to one end of known sequence, that prime in opposite directions and have a six-base cutter enzyme site (B) between them.
  2. e.g.        --------                          --------

    -------------------------------------------------=============

                |                          |                                              |

               A                        B                                             A

    ----- known sequence

    === unknown sequence

  3. Digest 1 microgram of genomic DNA with a 4-base cutter that does not cut between the ends of the two primers, or between the primer and the end of the known sequence. e.g. enzyme (A). Use 4-base cutters as the length of DNA must be PCR’able. Use several enzymes to be sure of obtaining a fragment of appropriate length.
  4. Heat kill enzyme 65 C, 20 min.
  5. Dilute to 500 ml with water, ligase buffer and add 10 ml T4 DNA ligase. Incubate at room temp for 2 hours. This will circularise DNA fragments as unimolecular ligation is favoured in dilute reaction conditions.
  6. Ethanol precipitate DNA, wash with 70% ethanol and air dry.
  7. Resuspend in 8 ml water. Add 1 ml of 10X restriction digest buffer and 1 ml of enzyme B. Digest for 2 hours. Heat kill enzyme.

The resulting fragment (the target sequence amongst the rest of the genomic fragments) looks:

--------                                          --------

-------------------=============--------------------

        |                                  |                                      |

       B                                A                                     B

and is ready for PCR.

Use 2 ml of the reaction mix from (6) in a 50 ml PCR reaction.

The unknown sequence (========) will be amplified.

 

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998