Why probe hybridizes to vector?
amarion at moose.uvm.edu
Tue Jul 11 13:43:43 EST 1995
faerber at ubaclu.unibas.ch wrote:
: In article <mic-1007952151470001 at 220.127.116.11>, mic at nwu.edu (Mic Chaudoir) writes:
: > In article <biolc4-020795165223 at mac-918.sr2-building.uh.edu>,
: > biolc4 at jetson.uh.edu (Wenfu) wrote:
: >> When I was using isolated DNA fragment( I am sure no any vector sequence in
: >> it)
: >> to probe digested plasmids, I found my probe hybridizes to the vector.
: > Whenever you gel purify something, a residual amount of other DNA in the
: > mixture comes along. For example, if one cuts out a single band from PKS,
: Further comment:
: for this reason we prefer to do a PCR with "insert-primers" and the insert as
: the matrix. The product is really only insert without any traces of vector DNA.
: This PCR product is then used as a template for i.e. Random Priming Labelling.
I routinely produce Southern probes by PCR using "insert-primers" to eliminate
the presence of vector sequences. However, I STILL see strong hybridization
of this probe to vector (specifically Bluescript) sequence. I believe this is
causing serious problems since I am trying to use this probe to screen a cDNA
library constructed in Lambda Zap II (ie., Bluescript).
marion at smtplink.ipfw.indiana.edu
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