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> From BIOSCI-REQUEST at net.bio.net Fri Oct 27 20:16 GMT 1995
> To: plant-ed at net.bio.net> From: c309scb at semovm.semo.edu (Dr. David Starrett)
> Subject: Hello and DNA isolation
> Date: 27 Oct 1995 12:31:21 -0700
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David Starrett writes:
>> Plant-ed compatriots,
>> I am a new subscriber to the group. Apologies if the following has
> recently been asked, discussed, etc. Does anyone have a good protocol for
> extracting DNA from any plant material which doesn't require centrifugation
> OR phenol, chloroform or other nsty chemicals (EtBr O.K.). Situation:
> University doesn't have a low speed centrifuge now. High speed not
> available for teaching. Teachin "lab" has no fume hoods, etc. I am
> teaching Plant Physiology to upper division students. They would like to
> extract plant DNA. I am trying to figure out a way to let them achieve this
> within the constraints of our equipment, etc. Any suggestions much appreciated.
>> Thank You,
>> David Starrett
> Biology Department, MS 6200
> Southeast Missouri State Univeristy
> Cape Girardeau, MO 63701
> Ph: (314) 651-2382
> Fax: (314) 651-2223
> email: c309scb at semovm.semo.edu>> The Science and Plants for Schools Programme (based at Homerton College
Cambridge, Royal Botanic Gardens in Edinburgh and Institute of Education at
the University of Wariwck) has developed techniques for safe and low cost
extraction of DNA from plants and subsequent restriction and gel
electrophoresis. The protocols are designed for use by school students (A
level - aged 16-18) but are also becoming more widely used in under grad
programmes.
We provide training courses for teachers who wish to use these techniques.
We use Sinapis alba cotyledons and grind about 40g in 5ml SDS buffer
(100ml buffer contains 73ml water, 10ml 1M Tris-HCl pH 8.0, 14ml 5M NaCl,
2ml 0.5M EDTA, 1ml 10% SDS) preheated to 65 degrees C. A little sand is added
to facilitate cell disruption. Transfer this to a small conical flask and wash
mortar with another 5ml buffer and add to flask. Cover with Parafilm and
incubate for 30-60min, agitating occasionally.
Remove debris by centrifuging at 2,500rpm in bench centrifuge. Filtering
would probably be OK for this. Put filtrate or supernatant into screw top
plastic tube.
Precipitate DNA by adding equal vol of cold 95% ethanol. GENTLE rocking of
the tube causes DNA to ppt over 5 min period. This can be centrifuged and
collected as a pellet or spooled out with st.steel wire bent to form a hook.
The DNA can then be dried prior to redissolving in TE buffer and restriction
by ether EcoR1, Hind 111 or Bam H1 (these are the enzymes we use).
Electrophoresis is on agarose gels poured in a low cost mini gel tank and
these are run overnight at 9v. Staining is by either Azure blue or methylene
blue.
We usually run plant DNA alongside lambda cut / uncut as standards.
RESULTS ARE EXCELLENT!
The materials and methods have been developed so that the technology is
accessible to schools on three counts:
low cost (very important in UK schools), safe, simple.
See:
Miller MB, Practical DNA technology in school. Journal of Biological
Education 28(3) pp203-211
Also contact National Centre for Biotechnology Education, University of
Reading, Whiteknights, Reading, RG6 2AJ Tel 01734 873743 Fax 01734 750140.
They market the kits we use.
Hope this might be of use to someone out there.
Best wishes, Barry Meatyard, Institute of Education, University of Warwick
Coventry CV4 7AL UK Email secab at csv.warwick.ac.uk
