I am trying to PCR amplify the complete mRNA (from first ATG to 3'UTR) from a human gene using genomic DNA. The total fragment has 3296 bp. I need the fragment to be until the end of the 3'UTR as this is where a SNP of interest is. My intention is to clone this fragment into a plasmid to do functional experiments. The fragment has 53.4% GC. I have tried to order the primers twice now in case there was something wrong with them.
I have tried the following enzymes:
- Bioline Immolase
- Bioline MyTaqHS
- KAPA2G Robust PCR kit and KAPA2G Robust HotStart (with both buffers A and B, enhancer...)
All these enzymes should be fine for fragments
Phusion enzyme worked fine for me till 4 Kb, I guess you will have to work on your PCR conditions. The 3'UTR region is in part in your Reverse primer, if I saw the sequence correctly, then are you sure your genomic DNA contains this SNP. As it is in the middle of the primer, if there is some mismatch, then the amplification might be a problem, with this long product.
Did you do any gradient PCR? Did you even see any faint band of your expected product?
Hii...Francine Z Marques,
Primers seems to be okay and GC content may not be problem. As you are getting non specific bands, its mainly because of misannealing of the primers..You can work on annealing temperature by gradient PCR from 55-65 range..Hope this will solve your problem...
Best wishes..
Hi,
We use KOD for just about everything we do in the lab and it works well for us-it is high fidelity and highly processive but will still work fine with long extension times (standard minute/kb you would use with other enzymes). You could extend the 5' primer a little just to balance Tms out.
Also DMSO or betaine as additives to your reaction might improve your signal but also introduce the danger of reducing fidelity-so you may get something to work with but it may not be 100% what you wanted.
What is the quality of your template like-can you obtain other PCR products from it? If not (and you have no other material) you could also try rescuing the template by using something like Genomiphi. I have used this in the past to rescue poor template and/or amplify limited template-it can help but not always.
Good Luck
Francine -
I always follow the calculation for optimizing the annealing temperature from the following paper: http://www.ncbi.nlm.nih.gov/pubmed/2243783
It takes into account both the primer sequence and the target amplicon sequence. But make sure to check the erratum published later that corrects the equation in the original paper. Erratum in Nucleic Acids Res 1991 Feb 11;19(3):698.