Genotyping Questions

[Jose de las Heras]

"Alexander Berkow" <alexbrkw at gmail.com> wrote in message 
news:mailman.1207.1159395447.20007.methods at net.bio.net...
> 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?

think about it: what is the *final* % of ethanol in the initial 
precipitation?

You added 100% ethanol, about 2.5 to 3 times the original volume, right? so 
the final % is about 70-75%

in other words: it doesn't change.


> 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?


nucleic acids absorb UV light especially strongly at around the 260nm mark. 
You can check it yourself if you have a scanning spectrophotometer.

the extinction coefficient varies for different species of nucleic acids... 
we normally use "readymade" conversion factors based on those coefficients, 
such as

Abs @ 260nm of 1  equals    50ng/ul of dsDNA
                                             33ng/ul of ssDNA
                                             40ng/ul of ssRNA

you can find all this stuff at the back of many catalogs, notably the NEB 
catalog (among many other reference sources, of course).


> 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?

I think you should check manufacturer's websites, and/or a good lab manual, 
such as good ol' Sambrook et al.


> 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?

are you sure?

PCR does not need loading buffer. Loading buffer is to allow easy loading of 
agarose gels, and is typically added after the PCR reaction.
DNA is already negatively charged, no need for an ionic detergent to provide 
a charge during electrophoresis.
Linearisation of DNA? I think you're confusing things... at least I am 
confused.

> 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?

electrons don't flow well through air ;-)

Jose
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