DNA extraction from very small volumes
Posted 11 May 2010 - 11:02 PM
I am new to this forum and have started working in a lab a few months ago. My project requires me to extract DNA from <100 uL of samples, and there might not be much bacteria in there too. Has anyone optimized very small scale bacterial DNA extraction before? What protocols would you recommend? Links would be very much appreciated
Posted 12 May 2010 - 04:25 AM
What is the nature of your small samples (e.g. stool, blood, sewerage, plant material)?
Are the bacteria therein viable? Can they be grown in vitro?
What will you use the extracted DNA for?
Posted 12 May 2010 - 04:54 AM
Posted 14 May 2010 - 02:26 PM
My samples are made up in mostly filtered water, and the bacteria is isolated from an aquatic environment. I am targeting the entire microbial community present in this sample, which includes bacteria and archaea.
Yes, they are viable but I probably can't culture them as I do not know the identity of the bugs in the sample and I wouldn't want any bias.. so I am looking to extract their DNA from this very small volume, PCR them and do pyrotag sequencing in order to characterize the community..
Edited by Phenol, 14 May 2010 - 02:33 PM.
Posted 16 May 2010 - 10:35 PM
edit: as far as I understood in your case the primers will be more decisive so to avoid bias I would use more primer pairs; the method for DNA extraction: use a standard protocol used in your lab or used in similar studies.
Edited by gebirgsziege, 16 May 2010 - 10:37 PM.
Posted 24 May 2010 - 09:37 PM