high-throughput DNA extraction

Dave.Edwards at bbsrc.ac.uk Dave.Edwards at bbsrc.ac.uk
Sat Nov 28 11:37:18 EST 1998


Patrick,

We extract from thousands of Mutator Maize plants for PCR screening
with the method below (not sure where it comes from). The grinding is
a pain but with plenty of liquid nitrogen it's fairly rapid. Let me
know if you come up with something better,

Thanks,

Dave Edwards

Dave.Edwards at remove.bbsrc.ac.uk
IACR-Long Ashton
Bristol
UK


1, With an eppendorf, trap 1/2/3 layers of leaf   and close the lid to
excise the leaf discs

2, Using the base of a blue eppendorf grinder, gently push the leaf
discs towards the base of the tube, then sit the grinder in the tube

3, Pour on an excess of liquid nitrogen  and grind to a fine powder

4, Place the samples in a new rack at room temperature to thaw

5, Add 500 ml extraction buffer  and vortex

extraction buffer
200mM NaCl
25mM EDTA
50mM Tris pH 7.5
0.5% SDS

6, Incubate at 65 oC for 5 minutes, vortex and reincubate for 5
minutes

7, Add 300 ml phenol/chloroform , vortex well and centrifuge for 10
minutes

8, Carefully transfer 400 ml of supernatent to a fresh tube

9, Add 400 ml Isopropanol and invert a few times

10, Centrifuge for 10 minutes, carefully remove supernatent leaving
the DNA pellet

11, Add 70% ethanol and centrifuge for 10 minutes

12, Remove most of the ethanol with a blue tip, centrifuge briefly

13, Remove the remaining ethanol with a yellow tip and air dry for 20
minutes

14, Carefully resuspend the DNA pellet in 100 ml TE

use 5 microlitres per 25 microlitre PCR


On 25 Nov 1998 06:29:22 -0800, Patrick S Schnable
<schnable at iastate.edu> wrote:

>We need to isolate DNA (for PCR) from many thousands of maize plants.
>We've found some very quick/easy extraction protocols that work on fresh
>tissues.  However, the big stumbling block is tissue grinding.  We would
>like to avoid the necessity of freeze-drying the tissue prior to
>extraction, but so far we haven't had good luck grinding fresh tissue in a
>paint shaker.
>
>Has anyone worked out a reliable, easy protocol from fresh tissue?
>
>Pat
>
>
>
>Patrick S. Schnable
>Professor of Plant Genetics
>G405 Agronomy Hall
>Iowa State University
>Ames, IA  50011 USA
>schnable at iastate.edu
>515-294-0975 (office)
>515-294-2299 (fax)
>http://www.public.iastate.edu/~schnable/
>




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