Double digestion and cloning - (Jan/25/2010 )
I am trying to insert a gene into clontech pTRE-tight vector (2.6kb).
For my GOI, I did the PCR on my gene (350bp) out from a plasmid template with the restriction sites at the end. I gel purified the PCR product and carried out double digestion with NEB EcoRI and BamHI. For the pTRE-tight vector, I also digested with EcoRI and BamHI, incubate at 37oC overnight.
I did the ligation and transformation. I get plenty of clones on my LB-agar plate. I chose 40 of them to propagate. After that, I redigested the miniprep clones with EcoRI and BamHI and check for the bands, I found that all of them are empty vectors (without insert).
Before ligation, treat the digested vector with Alkaline Phosphatase will greatly deduce the background.
are you sure both enzymes cut the plasmid? you can do single digestion to make sure. maybe one of them worked and the other one didn't, so when you ligated it actually religated to itself.
it would also be better to not gel purify the PCR product...just run a little of it on gel, if the band is there then purify the PCR product with PCR purification method in order to save more product. With gel purification you lose more PCR product