gateway cloning

StewJW via methods%40net.bio.net (by stewjw from gmail.com)
Fri Jan 9 04:38:07 EST 2009


Hi Koen,

I don't claim to be an expert in this area, but have you tried
linearising your  attB expression clones as described in the
Invitrogen manual. Also, have you purified the PCR product to remove
attB primers and any attB primer-dimers. Invitrogen also recommends
not to use phenol/chloroform extraction clean up methods. Like you say
its unlikely to be a size issue which is more of a problem with making
entry vectors using TA cloning methods where the upper limit is around
5kb. There is also something else nagging me about problems witrh
certain sequences, which is unpublished but I can't remember what it
is may be someone else can help here.

Best,
Stewart


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