Promoter insertion in pUAST vector - Promoter insertion in pUAST vector (Aug/19/2005 )
I want to insert a new promoter(by removing the old) in pUAST vector between P3' end and SV40 region.But the SV40 region should be retained.Its properties should not be lost during restriction digestion.
kindly check the link for the diagram of pUAST.
Kindly suggest me a suitable technique which would solve my dilemma.
you can do a couple things. Remove the the sequence via rest. digest with pstI and another enzyme of your choice within the polylinker. Check the pstI for digest in any other region of the vector, first.
Insert your promoter. You can anneal two long oligos with the promoter sequence and compatible ends for ligation or amplify it using PCR.
This is the quick and dirty explanation, but there are other options for preparing the vector.