In article <Patterson.1124484180B at hsdndev.harvard.edu>,
Patterson at cvlab.harvard.edu (Cam Patterson) writes:
>>I am curious about how people deal with cloning of large
>(over 10 kb) fragments. <snip>
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.
sbyarm at rulsfb.leidenuniv.nl