Troubleshooting Digest, ligation, transformation
Posted 17 March 2009 - 07:02 AM
I'm using a pCR4 topo vector. I have a G418 tag along with my insert. My original plan was to just cut out the insert and flip its orientation (both ends EcoRI). When I couldn't get that to work, we decided to just cut out the insert and religate the vector without it. I can't get that to work either!
At first I thought that my EcoRI wasn't cutting. But it looks like it does on the gel. I gel extracted the vector, added ligase, and transformed it. I am getting the same amount of colonies on my negative control plate (with no ligase) as I am on the ligation plate. Which leaves me to believe enzyme isn't cutting.. although it looks ok on gel. Suggestions anyone?
Posted 17 March 2009 - 07:24 AM
Posted 17 March 2009 - 07:33 AM
What other details do you need?
Posted 17 March 2009 - 07:41 AM
Or transform after ligation with trean enhancer to gain more colonies?
Posted 17 March 2009 - 07:44 AM
4.0 ul DNA (1 ug)
0.5 ul EcoRI
1 ul 10x buffer that came with enzyme
4.5 ul water
Edited by ChrissyFL, 17 March 2009 - 07:59 AM.
Posted 17 March 2009 - 07:47 AM
Try cutting 1µg with 0.5µl enzyme in a solution of 20-30µL could be helpful?
Posted 17 March 2009 - 01:50 PM
how big should your vector and insert be? I can see cut vector otherwise why would you have 2 bands? If i was you, i would try to digest less because the upper band (guess that is your backbone) is quite strong and might contain cut and uncut plasmid and then you would get results like yours...