double digestion problem - additional band after double digestion (Jun/14/2006 )

hi all,

hope someone can help me: i want to subclone (it's not my first subcloning i would like to annotate) a pcr fragment with BamHI ('5) and PstI ('3) restriction sites from a vector into the pQE30 vector. after religation and transformation and incubation i have made a plasmid prep with resulting clones. after that i have made a double digestion with both enzymes and the restriction buffer for BamHI as recommended by the distributor to verify if the plasmid contains the insert or not. by gel analysis i see the cutted fragment with correct size but two closely bands for the cutted vector.
i have faced this problem with two independent pcr fragments. i have checked the sequence of the pQE30 vector for BamHI and PstI restriction sites but the software shows only one site for each enzyme.
yesterday i made a single digestion with just the pQE30 vector and BamHI and have also seen two bands. interestingly after the second digestion with PstI i see a clear sharpe band for the linearized vector without any additional band. so it's very irritating!
any ideas????
thanks in advance!

Could it be a partial digest? Sometimes it happens. I would try these 3 different conditions:

- digest longer
- use less BamHI
- use more BamHI

I hope it can help a bit.

Raffaela

hi all,

hope someone can help me: i want to subclone (it's not my first subcloning i would like to annotate) a pcr fragment with BamHI ('5) and PstI ('3) restriction sites from a vector into the pQE30 vector. after religation and transformation and incubation i have made a plasmid prep with resulting clones. after that i have made a double digestion with both enzymes and the restriction buffer for BamHI as recommended by the distributor to verify if the plasmid contains the insert or not. by gel analysis i see the cutted fragment with correct size but two closely bands for the cutted vector.
i have faced this problem with two independent pcr fragments. i have checked the sequence of the pQE30 vector for BamHI and PstI restriction sites but the software shows only one site for each enzyme.
yesterday i made a single digestion with just the pQE30 vector and BamHI and have also seen two bands. interestingly after the second digestion with PstI i see a clear sharpe band for the linearized vector without any additional band. so it's very irritating!
any ideas????
thanks in advance!

I think Raffaela is correct. You can still purify away from the uncut vector using a gel purification of each fragment. If you just want to see if the insert was there. I think you have enough evidence that it is...