Fw: NEED A PROTOCOL FOR DNA ISOLATION FROM RICE SEEDLING
dbell at qnis.net
Fri Feb 2 12:24:03 EST 2001
> From: narottamd at 123india.com
> RESPECTED SIR,
> I NEED URGENTLY A STANDARED PROTOCOL FOR ISOLATION OF DNA FROM
> IF SOME BODY HELP ME I WOULD BE HAPPY.
I NEED A PROTOCOL FOR ISOLATION OF ISOZYMES FROM RICE.
> THANKING YOU ALL.
> NAROTTAM DEY
> NAROTTAM DEY
> PLANT MOLECULAR AND CELLULAR GENETICS SECTION
> BOSE INSTITUTE
> P1/12 CIT SCHEME VII-M
> CALCUTTA 700054
> WEST BENGAL. INDIA
We have an old, but very good book on isozymes that we have used as a basis
for most of our iso-enzyme work:
"Isozymes in Plant Biology" edited by Dougas E. Soltis and Pamela S.
Soltis. Dioscorides Press, Portland, Oregon. Advances in Plant Sciences
Series Volume 4. Theodore R. Dudley Ph.D., General Editor.
Our protein grinding buffer is 50mM Tris-HCl pH 7.8 with 15% surcrose or
glycerol and 0.1% 2-mercaptoethanol.
I would suggest for the DNA that you start off with a basic CTAB extraction
if you looking for a home-made do it yourself procedure.
What do you want to do with your DNA afterwards? amplification?
Are you wanting to do hundreds of isolations in a short period of time? If
so you may want to try some quick and dirty isolation kit. Kits are
usually faster timewise, but more expensive.
this is a modified protocol from Saghai-Maroof et al 1984. Proc Natl Acad
Sci USA 81: 8014 - 8018.
1. grind 75mg fresh plant tissue in 1 ml grinding buffer (50mM tris-HCl
pH 8.0, 0.7M NaCl, 10mM EDTA, 1% CTAB, 0.1% 2-mercaptoethanol)
2. Incubate at 65*C 1 h
3. Centrifuge and Keep supernatant.
4. Add equal volume (~750ul) chloroform-isoamyl alcohol (24:1). Mix well
5. Centrifuge and carefully remove upper, aqueous phase.
6. Add 500ul isopropanol and 50ul 2.5M Na Acetate. Mix well and incubate
@ -20*C ~30 min
7. Centrifuge down precipitated DNA. Pour off supernatant so as not to
8. Air dry pellet ~20min and then add 200 ul TE Buffer (10mM Tris-HCl pH
7.8, 1mM EDTA).
9. Incubate at 65*C until pellet is dissolved
10. Treat with an RNase
11. Add 500ul ETOH and 20ul 2.5M NaAcetate. Mix well and incubate @ -20*C
12. Repeat steps 7, 8, & 9.
13. To quantify and check the quality of your DNA - Run about 3ul of this
DNA prep out on a 0.8% agarose gel with molecular MASS standards (The only
ones I could find are from BioRad, no affiliation). Stain with EtBr and
quantify your DNA by spot densitometry of some kind. Good genomic DNA
should show up as a nice tight band high up on the gel.
14. The real test is to see if your DNA is amplifiable.
Hope this helps
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: dbell at qnis.net
phone: 559 453-3170
fax: 559 453-3088
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