I am having a problem with my cloning. I am introducing random T to C mutations during the procedure. Not sure what the problem is. Two areas of concern: overgrowth of E coli (aging culture?) or too long cutting gel band on UV. Has anyone seen this problem?

Can you be a little more specific about your issue. Which methods are you using? Which gene are you interested in? Etc.

Tony Rook said:

Can you be a little more specific about your issue. Which methods are you using? Which gene are you interested in? Etc.

Yes, I am using a Proof reading taq to PCR up my favorite gene (MFG) from cDNA our lab made. I gel purified the band with a Qiagen kit. Used TOPO TA cloning kit to insert the gene. Grew colonies O/N. Did a miniprep on individual colonies grown ON in liquid culture and sent them off for sequencing. (As I am writing this I am thinking I can send my gel purified PCR product in for sequencing. This will answer the question: is my speed/technique for cutting the band while on the UV caused problems.)

I had thought that perhaps the consistent (but random) T to C mutations could be a clue as to where my problem is originating - something that may be common knowledge that I haven't yet learned.

Your problem is not likely to be arising from aging E Coli cultures. More likely are errors introduced during PCR. Also possible is that the sequencing is mis-calling the bases. Double check the chromatograms to be sure the sequencing software has called the bases properly.

You might want to double check the integrity of the "original" sequence of your gene. Is it a GenBank sequence, or an EST? Are you looking at a different species? Poly morphisms occur quite frequently, so less important than the base composition is the resulting amino acid sequence. Translate your clones and compare to the original protein - if there are no AA substitutoins, you have nothing to worry about.

If you are convinced the mutations are random and are coming from the PCR, sequencing your PCR product is not going to help you any, as it will be full of mismatches.

Repeat the PCR using larger amounts of template and fewer cycles. Double check your nucleotide concentrations and your Mg+ concentrations to ensure they ar4e optimal for your Taq polymerase. Lastly, consider using a super high fidelity Taq, such as pfu-ultra or vent (tough).

As far as mutations coming during cutting out the band - I have had problems with this, but usually only in situations with exposed C or G dimers (like ends cut with NotI.) If you are worried about mutations induced by UV light, include 1-5 mM Guanosine in your gel and running buffer during electrophoresis.