FW: Purification of Genomic DNA

Deanne Bell dbell at fresno.ars.usda.gov
Tue Nov 9 12:57:54 EST 2004


Hi Sam

What TYPE of PCRs are you planning to do?  Here is why I ask:  if you
are amplifying satellites or other small segments of the genome using
sequence defined type primers, where you are going to get one fragment
amplified, there is fudge room for some degradation of your genomic DNA.

If, however, you are doing techniques such as AFLPs, RAPDs and ISSRs,
they require a very good DNA prep with very high quality genomic DNA.
These are techniques that utilize restriction enzymes, generate high
molecular weight, and/ or a large quantity of amplification products.
(for example, if you get lots of bands or fragments up to 3000bp in
length)

You are correct in being concerned that your preps are not coming out
within the normal parameters of other people in your lab, especially if
you are using the same tissues and the same protocols.  This sounds
mostly like a technique issue which is only overcome by practice - so be
encouraged and keep trying :-)   Also, the proof is really in the PCR
amplification.  If your prep amplifies, who cares what the A260 is or
what the pellet looks like!

Regarding A260 / A280 for DNA quantification - this is from my personal
experience thus it is only my opinion - there are lots of substances
carried over from a DNA prep that contribute to false A260 readings.

I prefer to run 2 - 3 ul of my DNA prep on a 0.8% agarose gel, along
with a set of standards for molecular mass (my set is from biorad and
has a 1000bp fragment with 100ng of DNA; a 700bp fragment with 70ng DNA;
a 500bp fragment with 50ng of DNA and a 200bp fragment with 20ng DNA).
Stain the gel with ethidium bromide.  I use our imaging system to do
spot densitometry in order to QUANTIFY my DNA - the brighter the band,
the more DNA.  The gel also allows me to see the QUALITY of the DNA.  If
you have a smeary lane, the DNA shows signs of degradation.  If you have
a nice tight band very high up on the gel, it is nice long chromosomal
DNA.

I must admit that I do not have a lot of experience with mammalian
tissues.  But I hope my input here has been valuable to your learning
experience.

Deanne Bell
USDA Agricultural Research Service
Crop Diseases, Pests & Genetics
9611 South Riverbend Avenue
Parlier, CA  93648
voice  (559) 596-2806
fax      (559) 596-2921
dbell at fresno.ars.usda.gov
 
 

>-----Original Message-----
>From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk] On
>Behalf Of Samantha
>Sent: Tuesday, November 09, 2004 9:13 AM
>To: methods at hgmp.mrc.ac.uk
>Subject: Re: Purification of Genomic DNA
>
>hi
>
>to answer your questions,
>1. my genomic dna was extracted from heart tissue of rats
>2. i would like to do pcr reactions for the downstream procedure
>
>how come i know the traditional procedure didnt work? well...i measured
the
>OD260 and OD280 and the genomic dna purified is out of the normal
range..i
>mean the ratio of OD260 and OD280 and the pellet precipitated during
the
>process was extremely large and it shouldnt be like that...(From the
>experience of my labmates) Thanks
>
>Sam
>

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