Crude Chlamy PCR prep for PCR

ellis at wustlb.wustl.edu ellis at wustlb.wustl.edu
Sun Aug 17 11:31:43 EST 1997

        I am posting this to the whole group because it may be of general
interest.  I have had the best luck with very crude Chlamy DNA preps for
PCR.  (I have never used this for chloroplast DNA, though, and can't
vouch for its usefulness there.)  This prep has been very good to me.
It is generally quite reliable in my hands, is not very labor intensive,
and generates enough sample for several PCR reactions.  It came to me
through Bruce Taillon and has its origins in the Jarvik lab.

Lisa Ellis

1) In sterile 0.5 ml tube, mix 70 ul sterile H2O
                               20 ul 20 mg/ml proteinase K
                               10 ul 10X PCR reaction buffer
2) Scrape about 0.5 cm^2 of Chlamy from a plate with a toothpick or a
        plastic inoculation loop.  Resupend in proteinase K mix.  This
        does not need to be a complete, uniform resuspension.
3) Incubate at 58 degrees 1 hour.
4) Transfer tube to 95 degrees for 1 hour.  I use a temp block with large
        holes partially filled with sand or glass beads.  The temperature
        MUST be greater than or equal to 95 to kill the proteinase K.
5) Cell debris will settle to bottom.  Leave it there.  (Quick spin is
        OK).  Do not use debris in PCR.  I store this at 4 degrees for
        up to several months.
6) For PCR, I use 10 ul of this prep in a 50 ul reaction.  I find that
        I need to use slightly more polymerase that I would with a plasmid
        template if I am to get consistent results.

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