I am trying to amplify mitochondrial as well as nuclear DNA from fecal samples but the success rate is very, very low. The reactions are failing miserably.
I would like to know what kind of Taq polymerase is to be used? I am using the normal Taq polymerase.
Also what other PCR components should be replaced in order to get a better success rate?
You may wish to see this article: Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106538/
I would use Phusion as the DNA polymerase of choice for this experiment!
We have been working in fecal samples of various primates. For amplification of DNA we observed best results by using Top Taq Kit (QIAGEN) compared to others.
Hi,
The Qiagen stool mini kit is what we use.
We have found variable success with particular brands of taq. Our best results have been from Qiagen's multiplex mix (even though we are not multiplexing). We do not normally use the Q solution, but BSA is often helpful.
It will depend somewhat on the length of the product you are targeting. Many Taq's have a specific range that they are more efficient at. Similarly the annealing temperature of your particular primers. Some Taq's have specific temperature requirements.
Beth Clare
Hey everybody
Thanks for the inputs and Faruku thanks for the article.
I will surely incorporate some changes suggested here.
I am using the QIAGEN stool kit for the extraction and trying to amplify DNA of primates(golden and capped langur) from their fecal samples (as I cannot get an access to their tissues or blood samples as they are Endangered under IUCN and scheduled I species under Indian Wildlife Protection Act., so you know all the permit issues).
Also my marker is a 300 bp mitochondrial marker, which should not be of much problem, but there is something I am messing up I guess.
I will try this changes and see what happens.
Many thanks again to all of you.
the new PureLink Microbiome DNA Purification Kit is doing a good job at depleting inhibitors. for the existing samples that already have inhibitors- try 10-100x dilution, or AgPath TaqMan (tough enzyme & rxn mixture), or BSA additive- i love it, dramatically improves qPCR efficiency
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