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Big cloning

Joseph C. Bagshaw jbagshaw at wpi.WPI.EDU
Thu Jul 14 08:32:49 EST 1994


In article <Patterson.1124484180B at hsdndev.harvard.edu> Patterson at cvlab.harvard.edu (Cam Patterson) writes:
>Fellow netters,
>
>I am curious about how people deal with cloning of large
>(over 10 kb) fragments. We have had a lot of discussion
>in our lab about this topic, and haven't reached any resolution.
>Perhaps it would be useful to have a discussion here regarding
>this matter. I would be curious to know what size fragments
>people have been able to clone, into what vectors, using 
>what cells, what ligase, special tricks, etc. Thanks.
>
>Cam 

In my lab we recently sub-cloned some Eco RI fragments out of phage 
into pBluescript II.  We mmade no attempt to purify the phage
inserts, since the cloning arms can't sub-clone.  We just digested
phage DNAs, phenol extracted, ligated into non-dephosphorylated
plasmid and transformed store-bought competent DH5 alpha cells
from Gibco/BRL.  We got more recombinants than we could hhandle.
In several of these routines, the Eco RI fragment that went from
phage to plasmid was 15-16 kb.  No problem.

-- 
********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.wpi.edu
Roadkill on the information superhighway.



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