PCR from genomic DNA

[Klaus.Matthaei at ANU.EDU.AU]

>In article <199404061949.AA00341 at wugate.wustl.edu> ,
>krishnan at BORCIM.WUSTL.EDU writes:
>>Could somebody tell me how to PCR-amplify a 3 kb piece of DNA from total
>human
>>genomic DNA or from total yeast DNA
>
>In addition to what others have said:
>
>If your DNA is very viscous, you should consider making it less viscous
>by some vigorous vortex mixing or by digesting with a rare cutting
>restriction enzyme that does not cut between your PCR primers.
>
>Good luck,
>
>Jim Owens

I cannot agree with the above Jim (no offense).

We have been PCR'ing genomic mammalian DNA for many years without the use
of pretreatments including PCR from single cells that are just lysed in
water and used directly.  This the basis of our cattle embryo sexing assay
done on the kitchen table on the farm whilst embryos are being transferred.
 Hrer however the target is small (400bp).

Our analyses on possible transgenic mice from very crude tail DNA preps
(proteinase K, SDS, EDTA o'night and isopropanol spooling of DNA) for
targets in the 1-3 kb range also are not treated in any way except for a 3
min 'presoak' at 94*C.  We have never needed to use restrictions or other
ways to break up the DNA.

The biggest problem occurs when you use too much starting material.  We can
easily see a single copy gene in 35 cycles from 10-100ng of this "dirty"
genomic mammalian DNA using a Corbett capillary cycler (Australian) and
'Supertaq' (from Stehelin Basel) (depending on the primer set).  There is
also no need to increase the length of time of denaturation or annealing. 
I use 5-10 seconds (in-tube time).  As far as primer sets go if "Amplify"
says that it will run then it always does (Thanks Bill Engels).

Of course if you feel you need to use restriction enzymes choose ones that
donot cut your target.  Indeed this method is very useful if you have two
very closely related targets that can amplify from the same primers. In
this case it is sometimes possible to remove one target by restricting it
before or after PCR.

Cheers, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
PO Box 334, Canberra, ACT 2600, Australia
E-mail: Klaus.Matthaei at anu.edu.au

"I'd rather a full bottle in front of me than a full frontal lobotomy"
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