legatek at hotmail.com
Sun Oct 1 14:43:43 EST 2006
Alexander Berkow wrote:
> Hello. I have a couple of technical questions regarding the methedology
> chemistry behind genotyping procedures:
> 1) During the initial DNA extraction (in this case from rat tail snips),
> why is it customary to use 100% and then 70%? I understand that the
> has a higher affinity for water and will dehydrate the DNA, but why not
> use 100% ethanol both times?
> 2) During DNA quantification prior to the PCR, why is 260nm used as the
> wavelength that the spectrophotometer is set to? Is it at this wavelength
> that DNA most efficiently disrupts the beam of light? And can this
> wavelength be used for all nucleic acid concentration assessments, or do
> different wavelengths need to be used for, say, RNA, double stranded or
> single stranded?
Assuming rat tail snips are the same as mouse tail snips, I have a much
simpler solution. After liberating the DNA from the tail in 500 ul lysis
buffer, spin down the hair and collect 400 ul of the sup in a separate
tube. Add an equal volume of isopropanol, mix and spin for 5 minutes.
Aspirate the supernatant and resuspend in 100 ul TE. Use 1 ul for PCR
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