ligation of BIG inserts
Dr. Duncan Clark
duncan at genesys.demon.co.uk
Mon Mar 30 03:20:37 EST 1998
In article <3517A276.DBF76072 at orion.rz.mdc-berlin.de>, Boris Jerchow
<bjerch at orion.rz.mdc-berlin.de> writes
>I'm having trouble ligating 10 to 20 kb inserts into Bluescript (sticky
>ends). I'd be happy if somebody could give me a hint about ligation and
>transformation procedures (any addition to standard ligation protocol,
>electroporation rather than chemical transformation, ...?).
>Many thanks in advance
It's probably not the ligation that is the problem but the capacity of
the E.coli for such a high copy vector with that size of insert. Reduce
your copy number by using pBR322 then if necessary go lower still ie
something based around pSC101. If that is still a no good then it could
be your insert is just lethal.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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