Inseparable insert in gel purification of the vector - (Feb/11/2010 )

Hi All

I have serious problem with gel purification.

I am trying to remove the insert-beta actin- from my vector-RFP ruby.However, for last three weeks such a problem emerged that even I do double purification there are some inserts in my DNA vector.

As a result of test digestions, we understood that restriction digestions work well.It is suprising that the QIAGen Gel purification kit was working well one month ago.We tried same kit last week with another unrelated vector, the same problem we have seen.I haven't changed anything from protocol and anything from solutions.

What do you think?
You can find the process in the attachment.I appreciate all kind of help.
Emergent replies please...
Thanks

Heyyy
Isn't there anybody having an idea?I am still struggling...

The insert bands on your gel look very overloaded with DNA, so the amount of your insert band may be quite small. Nonetheless it is troublesome. You could prepare you vector DNA by PCR rather than cutting plasmid. Then, DpnI treat the PCR product (ok in the PCR buffer) purify, and then cut with BamHI and XhoI.

You could also try running a long, higher % (1.5 - 2 %) agarose gel o/n at a low voltage to get a better separation of cut vector and insert.