Library screening alternatives?
Anton Scott Goustin
asg at cmb.biosci.wayne.edu
Wed Aug 2 18:51:33 EST 1995
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. The problem for me is lambda purification. 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 purification. 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 PCR product->plasmid.
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