southern enigma
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southern enigma
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?
William
Newsgroup archive
Response:
$$$$$$$$$$$$$ 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)
Newsgroup archive
Mart is dead right. As a general rule, if you want to make a clean
probe made by (eg) PCR, you must thoroughly wash out the gel tank (and
any associated buffer-recirculation tubing), use fresh (preferably
sterile) buffer for making and running the gel and (if at all possible)
don't run any other DNA on the gel other than the probe. If you must
use a size marker, put it in a track far from the probe track and take
great care in loading it. Cutting a band out of a plasmid (as above)
poses greater difficulties because it means you inevitably get a
fragment contaminated with vector DNA. The further you run the gel, the
less problematical this effect should be. Avoid the silly tendency to
run skinny little gels on postage-stamp sized gel kits. They cause far
more problems than they solve. Use a decent sized (midi) gel kit with a
nice thick layer of agarose. It'll still be cheaper than the 32P.
For even more fun you can cut out the gel slice containing the probe
band and run it further on a clean gel to eliminate more of the vector
contamination -- but this is advice more often given than followed.
Virtually everybody learns this the hard way.
Best wishes,
--
Chris Boyd
Newsgroup archive
While plasmid DNA contamination of the fragment appears to be evident from
the reported hyb results, I'm curious as to why *all* the lambda ZAP
library didn't light up like a "starry night" on the plaque lift- this
library vector is basically lambda with an internal Bluescript phagemid.
Hmmmm.
Cheers
Karl the hepB guy
Newsgroup archive
response:
$$$$$$$$$$$ Just a quick answer to this question; there are two
possible contributors:
* any plaque-lift is not uniform, i.e., some plaques are transfered
more easily than others which may be due to the irregularities of
the membranes.
* the amount of lambda phage in different plaques can be variable
(size variation/replicational differences)
...and the net result: hybridization signal variation
(with minimal intensity differences; however one would expect much
stronger signals to noise levels from true-positives!)
Mart (mspeek@ebc.ee in Estonia)
Newsgroup archive
Last update 05-Apr-2004, Rating Good of 0 votes.
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I have another Southern Blot enigma! I have used a 500bp DNA fragment as a probe to screen embryonic stem cells clones (for my knock-out projetct) which should get a Neomycin cassette and be positive with my probe if this cassette was inserted by homologous recombination. For the first screening I had 10 positive clones. Then I thawed these clones from the master plate and check again (the SAME conditions): they were positive again but the negative control was not very clearly "negative". So, I did it again and since then I´ve done it 3 times and my clones were not "positive" anymore. The strange thing is that the probe has always cross-reacted with a smaller neomycin band (after BamHI digestion) but now I cannot see this band anymore! That´s why I cannot say my clones are negative now (better said, that I thawed the wrong ones) because I don´t get the cross-reactiv band either and if I hybridize with a neo probe, the bands are there!
Is it possible that a probe changes its binding specificity???
I will be very grateful if you could give me some ideas!
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