I'm trying to amplify 16S rDNA from total DNA extracted from Ixodes ticks. Due to an error in the extraction process, some of my DNA concentrations are very low, in the range of 5 to 0.5 ng/uL. Furthermore, this is total DNA, and from what I've seen in the literature, 10% or less of this is bacterial, meaning the 16S copy number will be pretty low and I functionally have 0.5-0.05 ng/uL of template.
I have already lowered my annealing temperature, increased to 35 cycles, and increased the Mg concentration to 2.5 mM final concentration. I get very faint bands from some samples and no bands from others. I don't want to use a nested PCR because I fear that will compound the PCR bias and mess up the eventual sequencing.
What else can I do to improve my yield from PCR?
I am agree with Artur Bruzynski, Repeat DNA extraction procedure once again since low concentration will cause baised results. Other options such as concentrating the template, amplification and reamplification of PCR product, nested PCR but in your case it is not useful.
We have done genomic stuff in bacteria from ticks and the DNA samples are always crap. If you are getting bands from PCR using 16S primers and they are at the right size then you are getting what you want most likely. Use a larger volume PCR reaction, go as big as you can and then concentrate the DNA after clean up. For 16S sequencing you will need about 150 ng total in a 20 µl reaction. Also you can do multiple reaction at once and combine them and then concentrate the DNA using ethanol precipitation.
Sometimes, addition of several extra cycles makes a mirracle. You may try to increase number of cycles up to 40 or even slightly more. Also usage of a good thermostable DNA polymerase is recommended when amplifying crtically low template amounts. We are using a Kappa polymerase from Elizabeth Pharmacon that has a good price performance ratio.
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