| | Re: Arabidopsis DNA Extraction from Leaves
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
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).
750ul/sample—EB Buffer: 100mM Tris pH 8.0, 50mM EDTA pH 8.0, 500mM NaCl, 10mM beta-mercaptoethanol
250ul/sample---5M Potassium acetate
700ul/sample---50mM Tris, 10mM EDTA pH 8.0
100ul/sample--10mM Tris, 0.5mM EDTA pH 8.0
Materials and incubators.
Blue plastic pestles.
65C Water bath