Problem with non-radioactive hybridization to GC-rich DNA
goldberg at bms.com
Tue Dec 6 10:36:27 EST 1994
I have been using the Amersham ECL Direct kit for non-radioactive hybridization of a ca. 580 bp fragment to a cosmid library of a GC-rich (72%) bacterium. My problem is that the fragment binds strongly to the cosmid sequences (derived from a Streptomyces plasmid vector, also ca. 70% GC) giving me uniform hybridization to all colonies. I have confirmed this by showing that, even under
stringent washing conditions, there is strong hybridization to a cosmid fragment in Southern blots. I thought about increasing the stringency of the initial hybridization by lowering the salt concentration (they recommend 0.5 M MaCl) but Amersham said it wouldn't help much (you can't raise the temperature since the enzymes in the buffer will be inactivated). Hybridization to chromosomal digests of the DNA is not a problem; I see a single band in each case. Has anyone had a similar experience with the Amersham system and are there any recommendations how to solve this problem (aside from using 32P). Any other kits
which may not be so prone to this phenomenon?
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