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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
>Boris Jerchow

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....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288

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