lambda gt11 insert PCR

Stuart Brown SBROWN at GAES.GRIFFIN.PEACHNET.EDU
Fri Jan 13 10:39:19 EST 1995


In article <950105134607.6021cedb at thorin.uthscsa.edu>
HARDIES at THORIN.UTHSCSA.EDU writes:
 
>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? ...
 
>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.
 
I pick single plaques off a plate by coring with a Pasteur pipette and
then blowing out the tiny cylinder of agarose into 500 ul of SM.  After
vortexing (to break up the agarose plug) and waiting for about an hour
to allow the lambda particle to diffuse out, I use I ul of the SM as
template for a PCR reaction.  Using primers that match the lambda arms,.
I never have any problem amplifying my insert (small 200-1000 bp inserts)
 
 
Stuart Brown                       |   Plant Genetic Resources
                                   |   Georgia Experiment Station
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  SBROWN at GAES.GRIFFIN.PEACHNET.EDU |   Griffin, Georgia 30223-1797 USA
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