Cloning of 3 kb fragment - (Aug/20/2008 )
I am having problems cloning a 3 kb PCR product into pET29B in E. coli DH5alpha. I get transformants, but on single enzyme restriction digestion of the recombinant plasmid, I get a single band with size in between peT29B and pET29B+3kb insert. There seems to be a specific clevage of the insert. My question is can such a restriction occur in a restriction negative strain like DH5alpha. How can this problem be overcome. Can you think of any other reason why I may be getting such a wierd pattern. Thanking you in anticipation
I had a very similar experience.
In my case, I used BamHI for restriction, its star activity occured to my PCR product, and the short fragment was cloned into pET vector instead of my full PCR product.
When I changed into SalI, I could finish the cloning.
I don't know why this happens sometime, but I recommend to change the restriction enzyme for your cloning.
I had a very similar experience.
In my case, I used BamHI for restriction, its star activity occured to my PCR product, and the short fragment was cloned into pET vector instead of my full PCR product.
When I changed into SalI, I could finish the cloning.
I don't know why this happens sometime, but I recommend to change the restriction enzyme for your cloning.
I am also in trouble in cloning of 3kb to a 13kb vector, the R-gene to bacteria is Spec+, need your help
With regard to star activity, an enzyme like BamHI always cuts its recognition site, but can also sometimes exhibit star activity. Star activity is not so much a property of the enzyme as it is a property on the conditions the enzyme finds itself in.
Usually, star activity rears its ugly head when digestion conditions are suboptimal, thus the solution to this problem is not necessarily to swap enzymes, but to adjust the conditions under which the digestion is performed. There's a pretty good overview of this phenomena here.
What Restriction enzyme or enzyme combination are u using for the subcloning!! Chk whether they have compatible ends or not if using two diff enzymes.
Usually, star activity rears its ugly head when digestion conditions are suboptimal, thus the solution to this problem is not necessarily to swap enzymes, but to adjust the conditions under which the digestion is performed. There's a pretty good overview of this phenomena here.
Of course the star activity doesn't happen often. It happened to me only one time for 8 years' cloning. I guess I put the enzyme too much, and BamHI cut on GAATCC site instead of GGATCC site. There are four star activity sites of BamHI according to Takara catalogue. Anyway it can happen, and I still don't like BamHI.