problem with bi-enzyme cloning - (Dec/24/2007 )
I have a insert gene with SalI at N-terminal, XhoI at C-terminal with designed primers. (protecter for enzyme digestion designed 6bp in each primer) (3.1kb)
according to NEB suggestion, using buffer3 + BSA for digestion.
I digest them together with SalI and XhoI for overnight. same for the vector (7.5kb)
Run gel and cut out for purification. ligation 16oC for 10hours.
After transfromated the ligation into DH5a, there are 60-70 colonies. I checked 6, all of them are negative and the gel showed one 4-4.3kb band + one 15-16kb band after digested the products with salI/XhoI. I want to know what's the problem in my system. how can I get 4kb band?
Thank a lot
I would check more colonies. I would check all 70 colonies. Can you do colony PCR? With colony PCR you can check all 70 with little trouble.
However 70 colonies for a transformation is very low. Do you have a positive control plate?
Did you dephosphorylate the vector? What were the dephosphorylation conditions used? And how much vector was dephosphrylated.
In anycase, is there a need to cut the vector with both SalI and XhoI? Can you use only XhoI?
Hmm... just to confirm things, was the insert gel purified? Was said insert checked to make sure it was the right size?
However 70 colonies for a transformation is very low. Do you have a positive control plate?
Did you dephosphorylate the vector? What were the dephosphorylation conditions used? And how much vector was dephosphrylated.
In anycase, is there a need to cut the vector with both SalI and XhoI? Can you use only XhoI?
Hmm... just to confirm things, was the insert gel purified? Was said insert checked to make sure it was the right size?
Thanks
I cannot check 70 of them. Do you have the protocol to check them by PCR? I never worked on that.
I did not dephophoralate the vector because the negative ones are not empty vectors
I will try digest them one by one. I designed for correct insertion with salI and XhoI on each of sides.
the insert gel purified. But I think the size is a little less than what I expected (2.9-3kb instead of 3.1kb according to the agarose gel). this might be the problem. then I need adjust PCR condition.
Search for colony PCR on the forum. You will find much and many things on this topic, including protocols.
I am not sure what you mean by this. However do consider that both SalI and XhoI produce ends that are compatible to each other. Thus the vector can recircularise to itself (thus dephosphorylation of the vector should be something to consider). And the insert can go into the vector in both orrientations.
I forgot to mention in my last post, I generally avoid using SalI in my ligation strategy. I have found working with SalI troublesome. DNA that have cut with SalI sometimes appear to trouble ligating for unknown reasons. Others in the forum and in my lab have reported problems with SalI. The DNA cuts find but it sometimes just won't ligate.
Compatible ends = dephosphorylation absolutely required. That explains the 15 kb band - two vectors are ligating together. You should dephosphorylate even non-compatible ends to make sure that any incompletely digested single cut vectors do not recircularise. Try using different enzymes in your screen too - two or three that digest the vector and insert in a few positions - much more informative.