I'm trying to clone a 4 Kb insert into PNl1.1 vector (3 Kb). I'm digesting both the vector and the insert with KpnI & XhoI and using alkaline phosphatase for the vector only. Also, I'm doing the ligation with T4 ligase. After doing the cell transformation, I just get few number of colonies which don't have my insert. I've checked the transformation efficiency using undigested vector, and it was almost 100% efficient. Actually, I don't know what's going wrong?
Thanks,
Christine
check your ligation product in the gel if you see any faint band at 7kb.
yeah, there is a very faint band at 7kb. Should this band be isolated and used for transformation?
yes, you can do that but then its better you pool few ligations and run on get and then isolate the band. please make sure the cleanness of the plasmid. This is a very crucial issue. which is 260/280 ratio. between 1.6-1.8 , this largely governs if you will get positive clone or atall any clone. i guess with pure ligated plasmid DNA , you should have it in a week.
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