1) UV measurements at 260 nm (OD260) are valid for BAC, in fact BAC are
much smaller than human genomic DNA (human chromosomes are about
thousand times bigger than BAC) and OD260 is OK for human genomic DNA.
2) The biggest problem with the OD260 is the possibility of
contamination with RNA, which may lead to an overestimation of the DNA
concentration. Cellular RNA (mostly the ribosomal and the tRNA) are
usually removed by RNase treatment during DNA preparation. However, the
digestion is never 100% complete, there are small undigested pieces
caused by partially double stranded regions (remember the tRNA
structure?) etc. These small pieces, as well as the ribonucleotides
from the completely digested RNA, will still absorb very well the UV at
260 nm, and could be co-precipitated (after phenol extraction) with the
DNA. We use an alternative estimation of the DNA quantity by spotting
the DNA samples onto the surface of an agarose gel containing EtBr,
along with DNA control samples (standards). Our DNA standards are 200,
150, 100, 75, 50, 25, 10 and 5 ng/ul. The agarose gels are prepared and
stored in Petri dishes and are better when several days old (allowing
quick intake of the solutions). We spot 0.5 ul per sample or standard.
This technique greatly reduces the chances of DNA overestimation because
nucleotides and small pieces of RNA/DNA do not absorb or absorb very
little EtBr. This method allows quick (albeit not very accurate)
estimation of the DNA quantity which could be performed on hundreds of
samples without significant loss of time. Actually, for our lambda
phage sequencing (template size 50 kb) we found that two to ten times
differences in DNA concentration between the various samples still work
OK. The great advantage is that we can detect some problematic samples,
which look OK on OD260, but turn out to have little DNA when spotted.
For them only it may be worth to do electrophoretic verification of the
DNA quality.
Regards,
victor
www.alphadna.com
Tom Stelick wrote:
Does anyone know of a good way to quantitate bac DNA. I can run it on a
gel but this is a very inaccurate method. Is UV spec of this large size
DNA still good? Any suggestion would be helpful.
Tom Stelick
TJS11@cornell.edu
BioResource Center, Cornell University
157 biotech bldg.
Ithaca, NY 14853
(607)254-4857
http://brcweb.bio.cornell.edu