Trouble inserting PCR derived insert into vector - Trouble inserting PCR derived insert into vector (Jul/07/2008 )
Hi all,
I have been trying to clone a small PCR product with a Bgl II and BamHI site into a vector that is essentially pSP72 with an insert already in it. What I'm getting when I try removing the insert with Bam and Bgl are two large bands, both larger than the vector, with no relationship in molarity, along with a bunch of other crap. It doesn't seem to be a problem with either enzyme or with the purification process, and I've tried this twice so far. Any ideas as to what might be going wrong?
Thanks.
A gel picture would help
Anways, without that..
1. May be you have an insert bigger than vector.
2. Did you isolate bacterial genomic DNA too?
3. Cut some other vector with these enzymes you have.
What does your vector look like with single digest? If you get multiple bands when cutting with just BglII or BamHI you have additional sites you were unaware of. What do you see when you run the vector uncut? Do you get the typical three bands or do you see contamination there (this would tell you if you have genomic DNA)? As mentioned by cellcounter, a picture of a gel would help. What size is the empty vector? What size is the insert you are removing? I assume you are cutting with the BamHI buffer but what are your digest conditions? How much, how long, ect.