Glycogen v tRNA and OLD BONES

[Peter Gegenheimer]

In <9403291318.4d9781ff.LN1 at Lincoln.cri.nz>, SCRIMSHAWB%GRIEHFS at LINCOLN.CRI.NZ ("Scrimshaw, Brian J") writes:
>Can someone in netland explain the relative virtues of glycogen
>and tRNA as a carrier? Are they just as good as each other for
>sticking to DNA?

Should be equally good per se. The advantage of glycogen is that it is not a 
substrate for, nor an inhibitor, of nucleic acid modification enzymes. It does not 
enter electrophoresis gels, and it is higher mol wt. It does absorb in the 220nm
range, so a scan of DNA+glycogen will not look like pure DNA.

One point to always remember when using tRNA OR glycogen carrier: the material must 
be rendered nuclease-free by proteinase K digestion, phenol:CHCl3 extraction, and 
EtOH pptn.

>I'm trying to extract DNA from old bones and have been using
>glycogen as a carrier, with little luck, it may just be the DNA
>in the bones is too degraded or a protocol problem. I had no luck
>with silica and am trying a proteinase K approach with
>concentration in a microcon 30 after addition of glycogen, any
>advice appreciated.

First advice would be to add some DNA restriction fragments - 32P-labeled if you wish 
- to your initial bone sample, and see whether they are recovered. Proteinase K 
treatment (in pres of 0.1-1% SDS + 10-100mM EDTA) at 55-60 deg C is very reliable for
other sources. Make sure you aren't trapping DNA IN, or losing it THROUGH, the 
Microcon.


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