Why probe hybridizes to vector?

Amy Marion amarion at moose.uvm.edu
Tue Jul 11 13:43:43 EST 1995


faerber at ubaclu.unibas.ch wrote:
: In article <mic-1007952151470001 at 165.124.225.226>, 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.

<stuff deleted>

: > 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,

<stuff deleted>

: 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).

Any suggestions?

Amy Marion
marion at smtplink.ipfw.indiana.edu




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