Big cloning

Roger "Don't call me Aman" Morton sbyarm at rulsfb.LeidenUniv.nl
Thu Jul 14 14:01:05 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. <snip>
>Cam 

I managed to clone a 20kb insert from a pSL301 (invitrogen, high copy 
number) based clone into a 9kb plant binary vector (a pBIN19 derivative, 
low copy). I must agree with another poster who suggested that not 
isolating the insert of an agarose gel probably helps in these matters. 
In my case 
the insert was in a Amp resistant plasmid and the vector kan resistant. I 
ligated KpnI digested plasmids mixed together without gel purification in 1
-phor-all buffer. The vector was dephosphorylated. I transformed the 
ligation via heat shock (into DH5alpha) and pulled out one positive 
out of 20 minipreps. 
If you can organise to do your ligations this way then you can use quite a 
lot of DNA in the reactions and then electroporation is not necessary.

---------------------------
Roger Morton
sbyarm at rulsfb.leidenuniv.nl



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