Problem with double digest cloning - (May/23/2015 )
I have my promoter of 1.8kb with SacI(5') and KpnI(3') cloned in the pBluescript vector.I am trying to create a gene construct in the pCAMBIA1301 vector.I have double digested both the pCAMBIA1301 and the pBSK-promoter with SacI and KpnI.I cut out the 1.8kb promoter and the digested vector from the gel and purify it using gel elution kit and got a good yield.
Then when I give it for ligation overnight at 22C and transform it into DH5alpha cells,I see no colonies.I have tried it multiple times and using everything from scratch.But no colonies to be found.I also tried using Acc65I as the isoschizomer instead of KpnI,but still it failed.
What could be the problem? I really have less than a month as deadline to create my entire construct and am still stuck at the beginning.
- do you run any control or run other ligations? is your T4 buffer fresh? It has ATP inside and it's important.
- pBluescript is high copy number, the other one I'm not sure. Lowcopy number plasmids give less colonies.
- it could be your ligation, but at the same time it could be that one of your REs on the vector is not being digested well. I know you are seeing one band after digestion on the gel, but this happened to me a lot, and instead of double digestion, I did single digestion twice.
The most common problem is poor quality competent cells. Do a transformation control with very dilute plasmid.
Other problems that occur frequently include exposure to uv light during gel band cutting.
To add to Phage's comments, try to cut the band from the gel in few seconds.