From jillm rubens.its.unimelb.edu.au Thu Dec 6 17:50:31 2007
Date: Fri, 07 Dec 2007 10:56:29 +1100
From: Jill Maddox <jillmrubens.its.unimelb.edu.au>
To: Multiple Recipients of <angenmapanimalgenome.org>
Subject: Re: long term storage of cattle blood for DNA isolation
Dear Steve
The 8M urea RT long term storage option sounds very useful. What extraction
method do you use afterwards, and is it suitable for preparation of DNA for
screening high density SNP chips? Are there any references for this method?
Here is my 2c worth: In terms of FTA extraction, we have found that the
easiest is just to do a variant of the 20 mM NaOH extraction of Zhou et
al., (2006) A two-step procedure for extracting genomic DNA from dried
blood spots on filter paper for polymerase chain reaction amplication.
Analytical Biochemistry 354, 159-161. We do 3 mm punches and a single 30
min 200ul 20 mM NaOH wash, then a couple of rinses in T.1E (not sure
whether these are necessary) and then boil off the DNA in 120 ul H20 and
then use 2 ul per PCR (we dry this on PCR plates (65C, 15 mins) so we can
then just add small volume (at most 4 ul) PCR cocktails) . The method can
be scaled up with bigger punches and 96 well format using deeper tubes. The
DNA solution can be freeze thawed multiple times before use in PCR and the
DNA plates don't have to be used straightaway - can be used for at least a
couple of weeks.
Prior to FTA paper we used to just collect whole blood in EDTA tubes and
then put the tubes in a -20C freezer (manual defrost not automatic defrost)
if we were going to proceed to do the extraction within less than a month,
or a -70C freezer for longer term extractions. One gets good yields but the
colour can be a bit offputting (greeny-gray or red tinge) if the blood has
been at RT for a couple of days before freezing.
Regards
Jill
At 02:32 AM 7/12/07, Steve Kemp wrote:
>
>There are many options, and I am sure there will be other suggestions
>but:
>
>For small amounts FTA-paper works very well, although it can be tedious to
>recover DNA from many samples.
>
>You can put whole blood into equal volumes of buffered 8M urea. That will
>keep for ever at room temp. But the big volumes are a nuisance.
>
>But if you can spin and do a crude buffy coat separation, then dumping that
>into an equal volume of 8M urea will give you a much smaller and cleaner
>sample. In the field, we have used disposable plastic pasteurs to take
>around 0.5 ml buffy coat direct from a centrifuged 10ml vacutainer. That
>was dumped into approx equal volumes of urea in a 1.5ml cryotube. This is
>very quick and easy and samples last for ever at room temp.
>
>HTH
>
>Steve Kemp
>
>
>On 6 Dec 2007, at 00:02, James E Koltes wrote:
>
> > I am looking for a way to preserve blood samples (10-20mL) from
> > cattle for an extended period of time (1-4 weeks) prior to DNA
> > isolation.
> >
> > We are currently using purple top, EDTA tubes for blood sample
> > collection.
> > Then, we isolate the buffy coat using red blood cell lysis buffer
> > and PBS
> > incubation/ washes for downstream DNA extraction. This is not an ideal
> > situation, as isolating DNA sooner is not an option. Any
> > suggestions for
> > archival of blood or DNA extraction methods that would improve DNA
> > yield on
> > archived samples would be greatly appreciated.
> >
> > James
> >
> >
> > James Koltes
> > Interdepartmental Genetics
> > Iowa State University
> > 2361 Kildee Hall
> > Ames, IA 50011-3150 USA
> > Phone:515-294-9086
> > jekoltesiastate.edu
***************************************************************
Dr Jill Maddox Senior Research Fellow BVScHons PhD GDipAppSci(InfSci)
Faculty of Veterinary Science (Parkville) University of Melbourne Victoria
3010 AUSTRALIA Delivery address: Room G7, School of Veterinary Science
Corner of Park Drive and Flemington Rd
Parkville
Phone: 61 3 8344 5736 / 7344
Fax: 61 3 8344 7374
E-mail: jillmrubens.its.unimelb.edu.au
WWW: http://rubens.its.unimelb.edu.au/~jillm/jill.htm
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