I have a continuos problem regarding a double digest of a vector of 10kB where the restriction sites are only one nucleotide apart. I've tried double digesting (BamHI-HF and XbaI) but it didn't work and when I tried to ligate and transform I had many false positives. I had colonies but they were empty so it ligated back which says that the vector was not digested by both but only one. so, now I have three different vector digestions:
1) first with BamHI and then with Xba
2) first with Xba then with BamHI
3) both at the same time
I ran them on the gel and purified them. after phosphatase treatment I will ligate them with a PCR product.
I made a PCR and added restriction sites to those ends for Bam and Xba. is there anything I should be careful with when I digest the PCR product? should I double digest or not? Shoul I purify the PCR product before digestion?
any comments, suggestions are very welcome.