PCR from lambda library

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue Mar 29 17:24:14 EST 1994


In Article <9403292027.AA03689 at cardinal.ncsc.org>, mbwhite at CARDINAL.NCSC.ORG
(Martyn White) wrote:
>
>        Does anyone have a protocol for doing PCR from DNA extracted
>from a lambda library using a gene-specific primer?
>        Has anyone got this to work?
>
>Thanks in advance
>
>Martyn White
>  
   We do it all the time.  There are two advantages for doing cDNA work: 1)
you don't have to do a reverse transcription before doing your PCR and 2)
you are sure about there being something in the library to screen for once
you have confirmed that the PCR product is real.  As far as protocol, we
just prepare some DNA from a pool of the library using the Promega lambda
DNA purification kit and then PCR as per normal.
   One wrning: if you are using degenerate primers and/or low annealing
temperature, the lambda arms and residual E. coli DNA are a source of false
template.  Sequence of the products will of course tell you if that has
happened.
Good luck,

Warren Gallin,
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca



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