Genomic DNA Library - Problem of reactivity - Genomic DNA library screening with antibody (Jan/15/2010 )
I constructed a genomic DNA library of a bacterium using Partial DNA digestion employing Lambda ZAP II Kit (Stratagene). I did everything alright as shown in kit. I used 2-4 kb fragments for ligation with pre-digested vector supplied in kit. I used 1 uL of ligate for packaging and then amplified the library. In Blue-white screening, all the plaques showed transperant or clear with blue rings at the edges. There was no blue colony. I thought all are recombinant clones. Then, I started screening the library using antibodies. But I am not getting any positive clones. Please any one help me and tell the reason? My antibodies are good and I checked them several times.
isolate plasmid DNA from your library and check the plasmids for inserts by PCR or restriction digest.
Maybe your X-Gal is not working ...you can check that easily by transforming empty vector as a control!