Double digestion and cloning
Posted 25 January 2010 - 09:39 PM
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).
Posted 25 January 2010 - 09:54 PM
God helps them who help themselves!
Posted 25 January 2010 - 11:21 PM
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
Edited by Curtis, 25 January 2010 - 11:22 PM.