protocol for plant DNA isolation

[nanditajena]

i want to know the recent and accurate protocol for plant DNA isolation.

[danfive]

CTAB or Qiagen DNeasy Plant DNA extraction kit or similar from different company.

[yourlabdata]

I use a slightly modified edwards prep.

Much much much much easier than CTAB and much much much cheaper than quiagen kits... depending on how many preps you are doing extraction time can go as low as 2 minutes per prep and the dna is good enough to do preety much anything with... I havent done a quiagen kit in 3 years!

[yourlabdata]

Hi!

I use this protocol here:
Edwards Preps (A simple and rapid method for the preparation of plant genomic DNA for PCR analysis)

The pdf version of it is available here:
Edwards Preps PDF

Hope it helps!

[danfive]

Wow that Edwards protocol is extremely short. Some questions, how long do you expect your isolated DNA to last? Does it work on root tissue as well as leaves?


I've posted this protocol before, I used itfor gene mapping and produces stable DNA that would last 1-2 years at -20C.

Originally Posted by danfive

Here's a procedure I used in the past:

1. Weigh 0.1-0.2 grams tissue (~4 full trifoliate leaves) in 1.5ml microfuge tube. Quick freeze and grind to powder with blue pestle –don’t let thaw
2.Add 0.75ml EB buffer
EB buffer: 100mM Tris pH8.0
50mM EDTA pH8.0
500mM NaCl
10mM beta-Merc (add beta-merc fresh everytime)
3. Continue mixing until mixed well and thawed.
4. Add 0.05ml 20% SDS, shake well and incubate tubes at 65C 10min
5. Add 0.25ml 5M potassium acetate, shake well and incubate tubes on ice (0C) 20min. (Keep on ice until all samples are lysed).
6.Spin tubes at 21,000g for 20min
7.remove supernatant w/pipetman (~1ml) and put through mira cloth into new 1.5ml tube.
8. add 500ul isopropanol, invert to mix well and incubate at -20C for 30min-1hr.
9. pellet DNA at 21,000g for 15min—pour off supernatant and dry pellets by inverting tube on paper towel for 5min.
10. Redissolve pellets with 0.7ml TE (50mM Tris, 10mM EDTA pH8) by agitating; making sure pellet is resuspended.
11. Spin tube at max speed for 10min to remove insoluble debris.
12. Transfer supernatant to new tube and add 75ul 3M sodium acetate and 500ul isopropanol.
13. Mix well by inverting and spin to pellet DNA for 30sec.
14. Wash pellet with 700ul 80% EtOH—spin and remove EtOH, dry ~5min inverted on paper towel.
15. redissolve in 100ul 10mM Tris, 0.5mM EDTA pH 8.0.
16. Let sit ON at 4C.
17. Check OD260 and dilute for PCR (~1/100 or 5ng/ul).

Solutions.
750ul/sample—EB Buffer: 100mM Tris pH 8.0, 50mM EDTA pH 8.0, 500mM NaCl, 10mM beta-mercaptoethanol
50ul/sample—20%SDS
250ul/sample---5M Potassium acetate
700ul/sample---50mM Tris, 10mM EDTA pH 8.0
1mL/sample—Isopropanol
75ul/sample—3M NaAcetate
700ul/sample—80% EtOH
100ul/sample--10mM Tris, 0.5mM EDTA pH 8.0

Materials and incubators.
Blue plastic pestles.
-20C freezer
65C Water bath
4C refrigerator
Ice 0C
spectrophotometer
Mira Cloth

[yourlabdata]

Hi!!!

I still use dna that i extracted this way about 2 years ago and its fine... just make sure you use TE not water.

I have maped to genes using this protocol to extract DNA and have been able to, with practice, reduce the time per sample to about 2-3 minutes!!!

I strongly recomend it!!!

I have no idea if it works on roots.... Worth giving it a shot since its so quick!

Cheers,

Ed.