addition of BclI restriction site to PCR primer
Posted 22 February 2010 - 02:41 AM
I am trying to subclone an expression construct into a new vector in which one can only put an insert with BamHI - NheI. Since my insert has a BamHI (and BglII) site, I cannot use these enzymes to cut out the insert from the original vector. Instead I have designed PCR primers with a BclI restriction site in front and NheI at the end. I did the cloning in parallel with a colleague, the only difference being that her forward primer contained a BamHI site (and of course I did the digest with BclI at 50°). Her`s worked, mine didn`t, so I am suspecting now that it is something BclI related. I added 4 additional nucleotides on the primer next to the BclI restriction site as I always do. I could not find any information about whether this is to short or not. Does anybody know whether this might be the problem and how to solve it? I am also open for other suggestions to improve my cloning strategy.
Thanks a lot
Posted 22 February 2010 - 03:14 PM
BTW, according to NEB (see here), NheI only cuts 10% after 2 hours with three 5' bases... You could equally expect this enzyme to give you trouble.
Posted 23 February 2010 - 02:02 AM
Posted 23 February 2010 - 10:03 AM
Posted 23 February 2010 - 06:33 PM
check on the methylation sensitivity status of enzyme and the nearby sequence.
I think I saw that BclI was blocked by dam methylation -- but I chose not to mention it because you're digesting a PCR product (which won't be methylated). However, if you do go the intermediate plasmid route, scolix has a critical point -- be sure your recipient strain is a dam mutant.
Posted 23 July 2010 - 08:07 AM
are you sure your BclI enzyme can cut the site when you did not used any sequence before the restriction site?
I do not know Why new england biolab did not defined any sequence for this enzyme. I have to design a primer with the site for this enzyme but I am not sure it can cut the sequence
Posted 25 July 2010 - 11:40 PM