Genotyping Questions

Tom Anderson ucgatan at ucl.ac.uk
Thu Sep 28 07:32:43 EST 2006


On Tue, 26 Sep 2006, Alexander Berkow wrote:

> 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?

I don't know your specific protocol, but the point of 70% ethanol is
usually to remove low-molecular-weight salts (which are fairly soluble in
it) from the DNA (which is not). I'm less certain what the point of the
100% is.

> 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?

More or less - although it's absorbing the light rather than disrupting
it. See:

http://www.scienceisart.com/A_DNA/UVspectrum_2.html

I understand the maximum is actually 257 nm, but 260 is certainly close
enough.

> 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?

Yes, although the extinction coefficients are different.

> 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?

The minimal PCR buffer is a magnesium salt (usually MgCl2), as magnesium
is a cofactor for the polymerase, a mix of all four dNTPs, which are the
raw material and energy source for the polymerase, a buffering agent
(usually tris-HCl), to stabilise the pH, and an inert salt (usually KCl),
to give the right ionic strength.

To that, there is then a plethora of weird and wonderful additives that
you can use to improve performance if you've got trouble: ammonium
sulphate, DMSO, glycerol, BSA, gelatin, spermidine, betaine, formamide and
other small amides, nonionic detergents, PEG, tetramethyl ammonium
chloride, DTT, bME, oxalate and probably others. Sometimes they help,
sometimes they make things worse.

Tavi's PCR page is a really good source of info on PCR:

http://info.med.yale.edu/genetics/ward/tavi/PCR.html

> 4)  During PCR, the DNA loading buffer

Hang on - you you mean the buffer you use during PCR, or the loading
buffer you use after? Either way ...

> that is used is similar to SDS during an SDS PAGE assay, right?

No. There's no equivalent to SDS in DNA work because ...

> This molecule surrounds the DNA with an overall negative charge and
> causes the linearization of the DNA.

DNA has so many phosphate groups that at any but quite acidic pH, it has a
strong, uniform negative charge of its own, meaning it dissolves and
migrates in an electric field quite happily without assistance.

> 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?

Yes - and to stop the gel drying out! In principle, you could do AGE
without a buffer, if you attatched electrodes (strips of metal, say) to
the ends of the gel and wrapped the whole gel in clingfilm or something.
Indeed, Pharmacia make (made?) an electrophoresis rig called the
PhastSystem which, AIUI, worked like this.

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson at ucl.ac.uk



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