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Restriction site rearrangement - (Mar/26/2013 )

Hi guys,
I am using a p1006+ pHSV amplicon vector for my cDNA insert. In the MCS, I have the restriction enzyme sites in the following sequence:

- Asp718 - BamHI - EcoRI - EcoRV - XhoI -

flanked by a promoter and a packaging sequence. The problem is, I need the rest. enzyme sites rearranged so that EcoRV comes first, followed by BamHI. I need this the sites in this order so that I can put in my insert in the right direction.

What is the best, quickest strategy for obtaining a new vector that has these two sites in reverse order?



Design primers to amplify your vector with the correct RE sites in the 5' tail (followed by some bases that will be cut off). PCR your vector, cut with DpnI to remove the template vector, and purify. Cut with your vectors and ligate.