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problem in cloning involving partial digestion - (Mar/17/2012 )

Hello friends,

I have a plasmid of 10 kb size with two restriction sites for the same enzyme. If I try to digest this plasmid partially with the enzyme so that it gets cut at a single site, is there a possibility that one site is favoured over the other?

There is no option to find out which site is getting digested, as on gel, the two linearised fragments will appear as the same band. Pls shed me light becoz this is creating problem in my cloning experiments.

-sutanuka-

Dear sutanuka,

plz have a look at the following link ...it includes usefull information on partial digestion using Ethidiumbromide:
http://bitesizebio.com/articles/the-why-and-how-of-ethidium-bromide-assisted-partial-digests/

To check which site has been cut you need to perform a second restriction digest with an alqiquote of your already linerized plasmid ...the length of the fragments will then tell you which site was cut by the first enzyme.

Hope this helps!
Good luck!
Best regards,
p

-pDNA-