{NGS Group APS Sheffield} Downstream effects of carrier RNA in RAD/ddRAD

[Clemens Küpper]

Hi Jenny,

Moderate amounts of RNA are not a problem for RAD/ddRAD as far as I’m aware. The issue might perhaps rather affect the accuracy of your DNA quantification, e.g. the nanodrop will give you a combined estimate of nucleic acids (RNA&DNA). Using an assay that is DNA specific (Hoechst Dye w Fluorometer or Promega’s Quantiflor Picogreen) can help you to get around this problem.

I’d be curious to know more about your protocol in regards to the old feather samples. The DNA must be severely degraded which should affect your ddRAD libraries. How do you get around this problem?

Cheers

Clemens

--------------------------------------------

Clemens Küpper

Department of Animal and Plant Sciences

University of Sheffield

Sheffield

S10 2TN

UK

Phone: +44 114 222 0106

[Jenny Kaden]

Hi Clemens!

Thanks for the info. If we need to use the carrier RNA we'll just go ahead and use it then. Good to know someone has some information about this! (I couldn't find a thing on the internet).

We quantify the DNA for ddRAD using a QUBIT (and check the quality on a gel). Although in general it is very important to have good quality DNA going in to the ddRAD library, I have also heard that if you have to use some degraded samples, as long as all your samples are of similar quality/quantity, then you can still get some useful information out of the library (where as if you add poor samples to an otherwise good set of DNA, they tend to get drowned out by the better samples). So fingers crossed that A. We get some decent DNA, or B. They're all the same quality and something will work!

Thanks for you help (and quick response!),

Jenny

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