john.brennand at gbapr.zeneca.com
Tue Jun 11 05:57:22 EST 1996
Here's my twopenneth
It is easy to check whether your ligations are working - set up
insert DNA alone
vector + control PstI fragment
vector + insert DNA (vary the ratios, 1:1, 1:3, 1:10)
All +/- ligase overnight.
Run 1/3 of the DNA's on a gel and see whether the patterns are as
expected. If they are not, I would suspect that the long PstI
incubation has "nibbled" the ends - use more enzyme for less time.
If they look OK - transform. If you dont see any colonies and the
transformation control is good I would suspect that the insert DNA
is being restricted by residual enzymes in the host strain -
therefore try alternative host strains to transform.
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