Analyzing transformants

Jose de las Heras josenet at tiscali.co.uk
Thu May 4 14:21:41 EST 2006


By PCR *and* digest? why both?

you can find a restriction site close to one end in your insert, and either 
with that alone or in combination with another in the vector you should 
generate a pattern that will tell you in which orientation the fragment is.

But it's easier by PCR alone, if you can wait to get the primers, one from 
the vector and another from the insert, as others explained. It's the 
easiest way...

Jose

"newsnet customer" <customer at newsnet.com> wrote in message 
news:ZQk6g.22547$vy1.3229 at news-server.bigpond.net.au...
Hi

I have transformed GFP gene into a vector.
Then transformed the vector into competant E.coli cells.
I have many colonies.

QUESTION:

How to check if colonies have my gene in the CORRECT orientation by PCR and 
restriction digest? I think the only way is to sequence the region in both 
directions.

Any comments appreciated.

Cheers,
ST




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