Genotyping Questions

[Alexander Berkow]

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?

3)  Besides MgCl2, what else is present in the Taq buffer used in the PCR
reaction?  Salts, I presume, pH balancers... anything else?  ATP for the
polymerase?

4)  During PCR, the DNA loading buffer that is used is similar to SDS during
an SDS PAGE assay, right?  This molecule surrounds the DNA with an overall
negative charge and causes the linearization of the DNA.  So... what is this
molecule?

5)  When running an agarose gel, why is a buffer required as a bath for the
gel?  Is it just to provide electrolytic ions as a substrate for the current
to pass through the solution?