Genotyping Questions

Kyle Legate 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 
> and
> 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 
> ethanol
> has a higher affinity for water and will dehydrate the DNA, but why not 
> just
> 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 
analysis.

Uncomplicated.


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