Library screening alternatives?

[Steven Sullivan]

Anton Scott Goustin (asg at cmb.biosci.wayne.edu) wrote:
: sullivan at gwis2.circ.gwu.edu (Steven Sullivan) wrote:
: >
: >
: >It's been some years since I screened a lambda gt11/cDNA library, using
: >the tried-and-tru (but labor intensive and slow) plaque hybridization
: >method -- I'm wondering if in the interim any cool new (e.g. PCR based?)
: >methods have arisen to make library screening quicker, easier, etc. 
: >
: I don't find screening to be a drag.  It's rather fast if you have a good hot probe, 2 days from plating to picking the plaques.  Th=
: e problem for me is lambda purification. 


Well, I meant the *whole* process, including the dreaded plaque 
purification step.

 That takes time, especially if you like crowded plates (50,000 plaques
per 150 mm plate), = : which requires secondary and tertiary
purifications, and then--DREAD!--growing up a good-sized high-titer phage
stock for DNA purifi= : cation.  Remember, to get 1 µg of 2 kb insert you
need 20 µg of pure recombinant DNA.  I find it very easy and convenient to
pop out= :  the inserts using PCR.  New England Biolabs sells forward and
reverse primers for lambda gt11 flanking the Eco RI site (24-mers).  = :
They work easily, especially if the insert is not cut by Eco RI, allowing
a very quick shuttle from phage->PCR product->EcoRI-cut PC= : R
product->plasmid. 


do you worry at all about fidelity of the PCR product?