mspeek at tamm.eenet.ee
Thu Jan 22 06:55:07 EST 1998
William Sun (william at neuro.usc.edu) wrote:
: I have a 600bp DNA fragment which I cut out of a plasmid, purified from agarose
: gel with Gene Clean and labeled with 32P using random prime method. I then
: used the probe to screen a lamba ZAP cDNA library (Stratagene) and
: isolated several clones. I isolated the clones and cut the inserts out for
: a southern blot. I then hybridized the blot with the same 600bp probe. To my
: surprise, I don't see any signals on the inserts. Instead, I see signals
: representing the 3 kb Bluescript vector and the bands in the 1 kb ladder!
: Anyone can think of an explanation?
$$$$$$$$$$$$$ The problem you encountered here is very simple and common!!!
(although I must admit not all mol-biologists believe that this is the case!)
Anyhow, when purifying your fragment from agarose what you get is not
only 600bp fragment, but there is always a backround smear which
includes small amounts of vector sequences (depending on the quality
of the plasmid prep!). After cutting out and amplifying, these
sequences will get labelled along with your fragment of interest.
That's why you end up with two different types of sequences which,
as a probe, are represented in excess in your hybridization.
You might also want to know that in BRL 1 kb ladder 1.6 kb fragment
and some smaller ones are derived from pBR322; According to the described
scenario these bands should, in theory, light up (as you described!)
Hope this helps!
Mart (in Estonia)
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