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addition of BclI restriction site to PCR primer - (Feb/22/2010 )

Dear all,

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

-Ecolina20-

It might be related to the BclI not cutting, then again, it might have just been a bad cloning day for you...:lol:. If you think you're having an issue with insufficient 5' bases (I couldn't find any info on BclI, either), you could clone your product into an intermediate TA vector, and digest it from there with BclI and NheI.

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.

-HomeBrew-

Thanks a lot, I think I just give it another go with new primers with longer extensions and hope this solves the problem.

-Ecolina20-

check on the methylation sensitivity status of enzyme and the nearby sequence.

-scolix-

scolix on Feb 23 2010, 01:03 PM said:

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.

-HomeBrew-

Hi
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 <_<

-vahid-

I did actually use a sequence of 4 nucleotides before the restriction sequence, but it seems to have been to short. In the end I designed new primers with an extra 6 nucleotides and then the cloning worked fine.

-Ecolina20-

It is check on the methylation sensitivity status of enzyme and the nearby sequence. 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.

Term Papers

-albertwilson-