purification、ligation and transformation problems - (Mar/16/2008 )
Hi,
I am convinced with Anwar. You can do one more thing of taking more DNA for initial digestion. By that way you will have more DNA after purification losses. In how many microliter of TE/ddH2O you are extracting ur DNA from gel?
Best luck...
-Niraj-
QUOTE (anwar_mt @ Mar 17 2008, 05:13 PM)
I spent almost 4 months trying to clone a fusion gene and always ended up with either no clones or few clones missing alot of my gene. But when I minimized UV exposure, Screening was almost 100% positive with realy high transformation efficiency.
According to your controls, it seems to me purification problem as you said.
In control number 1 you are not supposed to get anything and that's fine
control 2 worked because there is no gel purification
control 3 showed nothing because of the purification.
Try what I said, just cut the bands as fast as you can from the gel. No need to cut the band exactly, little bit of gel won't affect your purification. I used Qiagen in my purification and I think promega is good enough.
Give it a shot and let me know how it goes.
UV affects the ends of your fragments that'swhy they might not ligat.
Good luck
According to your controls, it seems to me purification problem as you said.
In control number 1 you are not supposed to get anything and that's fine
control 2 worked because there is no gel purification
control 3 showed nothing because of the purification.
Try what I said, just cut the bands as fast as you can from the gel. No need to cut the band exactly, little bit of gel won't affect your purification. I used Qiagen in my purification and I think promega is good enough.
Give it a shot and let me know how it goes.
UV affects the ends of your fragments that'swhy they might not ligat.
Good luck
Hi，
I did just as you advised. Yes, I got my clones! I appreciated you very much.
Best wishes!
-chunmei zhuang-