Quntifying small amounts of DNA reliably
natalieB at qimr.edu.au
Tue Feb 4 02:06:51 EST 1997
: mmd280 at sysc.abdn.ac.uk (s.pritchard) writes: > Hi
: > I am working on LOH in lung cancer and have to use paraffin embedded tissue
: > as the source material.The DNA is recovered from the tissue after standard
: > Haemotoxylin/eosin staining .PCR is performed on this material with no further
: > purification so as to minimise further degradation.The yields are obviously
: > small and therefore I can not estimate the conc. by running it on a gel and
: > i presume that such crudely prepared DNA would be useless for analysis on
: > a spec. Any ideas?
: > Thanks in advance
: > Stuart Pritchard
: > mmd280 at abdn.ac.uk
: Stuart, I've used an Ethidium Bromide Assay where you spot a 1 ul aliquot of your
: sample and also a 1 ul aliqout of a 1/10 dilution of your sample on a petri plate
: that contains 0.8% agarose and EtBr. You also spot a set of standards of known
: concentration (100, 75, 50, 25, 10 ng/ml). Let the spots dry for 10-15 minutes
: and then photograph the plate under UV light. Then all you do is compare your
: unknown to the standards. The agarose/EtBr plates can be prepared ahead of time
: and stored in the fridge for one month. It may be worth a try. Mike.
: Michael E. DeGraaf
: Pharmacia and Upjohn Inc.
: E-Mail: MEDEGRAA at AM.PNU.COM
I suggest you run your PCR with the same primers on pure DNA, and you
test serial ten fold dilutions of the pure template and
you compare the intensity of the bands to those you obtained
with the fixed tissues. It should be fairly simple. However, I don't
see the relevance of quantifying DNA from fixed sections.
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