help regarding starting concentration of dna

[balaji_chattopadhyay]

Hi,
This is a problem I have stated elsewhere as well. I am working with genomic DNA of mammals. I am trying to standardize RADTAG protocol. In this regard I need to know the importance of genomic DNA concentration for restriction digestion and the least quantity of genomic DNA that can be used. I use Qiagen DNA extraction kit and my samples have typically shown ~ 10ng/ul concentration in nanodrop. But flourometer (Qubit), returned genomic DNA concentration of~2ng/ul. The radtag protocols available usually refers to starting quantity of 100ng to 1ug, with most reports suggesting 1ug. At the most I can try to concentrate my DNA and may obtain ~50 to 100 ng yield for a 50ul restriction digestion experiment.
It would help me much if I can get some advice regarding possibilities of standardization. My samples are obtained through non lethal sampling of wild caught animals and it may not be possible to obtain more tissue or repeat sampling.

[bbentlage]

Hey there!

I haven't done RAD tags myself but I have two comments that come immediately to mind.

1) Personally, I would trust the Qbit more than the Nanodrop considering how concentrations are measured i the two different technologies.

2) If you must "make" more DNA and cannot get more tissue samples you could look into something like the Genomiphi kit. Essentially you amplify the DNA in your sample using random hexamers and a proof-reading tag.

-Basti