Bisulphite Sequencing of clones but getting several products - (Mar/19/2008 )
I was wondering if anyone can help? I am currently sequencing clones from bisulphite modified DNA and having some problems as I am getting several different products in my sequencing results.
Briefly what I did was amplify my region of interest by using primers I designed myself, cut the band from the gel and then purified the sample. It was then transformed into E.coli and colonies picked the following day which were incubated overnight in LB broth and the DNA obtained by plasmid prep. This product was then used for sequencing PCR using standard M13 primers and cleaned up using AutoSeq G-50 columns and ethanol precipitation before adding the formamide to run on the sequencer.
The products that I am getting all begin with the surrounding vector, followed by my initial forward primer, then a product (varying in length between 164 to 474 base pairs), followed by my initial reverse primer and surrounding vector. My initial thoughts are that my primers aren't specific enough but since I'm cutting the band from the gel I don't understand how I am getting such a range of sizes of products produced? Any suggestions welcome!
we had the same problems in the beginning and found an easy solution by using hemi-nested PCR i.e. use one of you external primers for the internal amplicon amplification. Another tip: use a reverse primer (or reverse M13) to do the sequencing, for some unknown reason this gives better results than using forward primers.
Hope this helps,
PS. probably unnessecary to say but make sure you do PCR gradients for each PCR.
i don't understand why you run pcr on a gel first - do you get so many bands after pcr that you really can't avoid this step?
If you use standard etbr+uv visualisation remember that it is very harmful and nicks DNA - this could be the reason why your products have various lengths.
another thing - are you sure that what you cut out of a gel is a single band, not a slight smear?
Just a word of caution with gradient PCRs, pick the amplicon of the highest Tm, because then you are sure that the primer is specific for fully converted template.
But also be wary that biases toward methylated templates could occur given the high Tm.
This is not to say that it shouldn't be done, but just to be aware.
Good luck !