lambda gt11 insert PCR

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Jan 5 14:45:48 EST 1995


John Aris writes:

> 	We are interested in using PCR to amplify inserts from lambda
> gt11, and are familiar with doing this using purified lambda DNA as
> template.  Can inserts be amplified without purifying the lambda DNA?
> If so, and suspensions of lambda phage particles in SM buffer can be
> used in PCR, how is this done?  How much plate or liquid lysate is
> needed? ...

I haven't done this, but here is some information that may help:

1)  Since the heat will denature the lambda coat, I think you can
    assume the DNA will be 100% available.  So if you know how
    much DNA you would have gotten from an aliquot of phage lysate,
    then you should be able to use the lysate as if it were that
    amount of DNA.

2)  Lambda is ~2x10^10 pfu/ug of DNA contained.  So if you have
    titers for phage stocks, you can convert to DNA concentration.

3)  30 cycles of PCR should be able to recover a signal from
    picogram amounts of lambda DNA.

4)  Liquid lysates should contain 0.1-1 ug/ml of DNA; plate stocks are
    presumably more concentrated.  So considerable dilution is indicated.

5)  SM is 10 mM Mg; the PCR rxn should come out to be 1.3 mM Mg;  Don't
    put a big slug of SM into the PCR rxn.

6)  A single plaque contains ~10^7 pfu or ~0.5 ng of lambda DNA.  I
    recall discussion in this newsgroup about people getting enough
    DNA out of the plaque with a toothpick to support PCR.

Hope this helps.
Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu






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