Library screening alternatives?
sullivan at gwis2.circ.gwu.edu
Wed Aug 2 19:11:12 EST 1995
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
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
do you worry at all about fidelity of the PCR product?
More information about the Methods