Lambda gt11 insert PCR

Clemens Peterbauer clemens at eichow.tuwien.ac.at
Fri Jan 6 09:54:34 EST 1995


In article <1995Jan4.145054.1 at icbr2.ifas.ufl.edu>, aris at icbr2.ifas.ufl.edu says:
>
>Dear Colleagues,
>
>        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?  Is a published method(s) available?  Related comments and 
>anecdotes would be appreciated.
>
>Thanks,
>
>John Aris
>aris at icbr.ifas.ufl.edu
>
>

Hi, hope this helps - did it myself successfully several times:

You can use a phage suspension as template, basically no matter how you got it - 
just eluted from the topagarose plug, from a plate lysate, or a liquid lysate. The higher
the concentration, the better. If you get 100.000 or more phage particles for your 
PCR rxn, it should be enough. Use as little suspension as possible to get the right 
amount. If you have to use more, make sure to include the amount of Mg in the phage
buffer into the amount necessary for the rxn - use a polymerase buffer without Mg, and 
add the proper amount of Mg separately (once I had to use 10 ul template in 50 ul rxn vol,
that makes 2 mM Mg, so I didn´t add any more - it worked)
Boil the phage particles for a few minutes to release the DNA. I usually added nucleotides 
primers and polymerase after a boil of 5 min. 
I got good results with 35 cycles. 

Good luck,
Clemens



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