cloning - help!!!!! hello people! we have a cloning problem in our lab and hope someone else out there may have come across something similar/some ideas (if this ng is inappropriate - sorry!). OK, we are trying to clone a 2kb fragment into a

Dom Spinella dspinella at chugaibio.com
Fri Mar 27 18:11:51 EST 1998


> 
> hello people!
> 
> we have a cloning problem in our lab and hope someone else out there may
> have come across something similar/some ideas (if this ng is inappropriate
> - sorry!).
> 
> OK, we are trying to clone a 2kb fragment into a 9kb vector (of our own
> construction based on bluescript). The 2kb fragment is a BglII fragment;
> it is treated with Klenow in presence of AG. The vector is linearised
> with XhoI (when run out shows single 9kb band) and Klenowed in the
> presence of CT. When these are ligated and transformed, we consistently
> get *only* a 3kb product. In addition, a primer pair (forward primer in
> vector, reverse in fragment) shows a positive result on this product.
> 
> We have controlled for everything we can think of - bacterial
> contamination, ligase, klenow etc.... but nothing has changed the fact
> that we only get this 3kb product instead of something around 11kb.
> 
> SO, has anyone seen something like this before, and/or got any ideas????
> 
> all help appreciated, all flames not.
> 
> frustrated of London

Dear frustrated:

Assuming you have ruled out trivial explanations, its important to note
that not every DNA sequence is clonable in plasmids. Sometimes the
sequence encodes toxic products for example. Bugs also don't
particularly like replicating sequences with a lot of repetitive
elements or large palindromes.  That's a big reason why there are gaps
in fine structure genomic maps derived from BACs or YACs for example. 
Sounds to me like your bugs are unhappy with your new 2kb fragment and
are rearranging the entire plasmid and deleting what isn't necessary. 
This is, of course, a rare event -- but you are selecting for it because
that's the only thing that will grow!  Happens all the time. Have you
sequenced your 3 kb plasmid through the presumed cloning site?  That
should let you know if and where the deletion is occuring. If this is
indeed what's going on, you can sometimes get around it by cloning in
lytic bacteriphage lambda rather than a plasmid.  I don't know if that
would help you, but there it is -- my guess about the problem.
--Dom Spinella



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