GENOMIC DNA EXTRACTION
(Adapted from: Doyle J.J. and Doyle, J.L. (1990) Isolation of Plant DNA from Resh
Tissue. Focus 12: 13-25)
DNA EXTRACTION PROTOCOL – PLANT TISSUE
1. Grind tissue under liquid nitrogen until tissue is like a powder, transfer into
sterilized eppendorf tube.
2. Add CTAB buffer with BME (1.0 ul of BME per 1.0 ml of CTAB), CTAB used
at 2.0 mL per gram of tissue. If using 0.1 grams of tissue, use 500 ul CTAB and
0.5 ul of BME.
3. Incubate extracts for one hour at 65°C – shake the samples every 10 minutes to
ensure that things are staying well mixed.
4. Spin at 13,000 rpm for 1 minute then pull off the supernatant into a new tube.
5. ***Can do multiple extractions of the same pellet and combine supernatants to
get a better extraction. If you want to extract again, repeat from step 2.
6. The combined supernatants are then extracted with one volume of chloroform.
7. Tubes are spun at 1000 X G (RCF) for 5 minutes (4°C)
8. Pull off the aqueous layer and transfer to a new tube.
9. To get more DNA out you can also “back extract” the chloroform with one
volume ddH2O. The “back extracted” tubes are spun at 1000 X G (RCF) for 5
minutes (4°C) and the aqueous phase (top layer) is transferred to the same tube as
in step 8.
10. Next the DNA is precipitated with 2/3 volume of isopropanol. The solution is
allowed to incubate at –20°C for at least an hour and up to overnight.
11. Tubes are spun at max speed for 5 minutes and the supernant is discarded.
12. Pellet is washed with 500ul of 80% ethanol the spun at max speed for 5 minutes.
13. Discard supernant and use a small (0.1-10 µL) pipette tip to pull out as much
liquid as possible.
14. Dry pellets by either leaving the tube open in a safe location or by using the
speed-vac.
15. Once pellets are dry, resuspend with 50-100ul of 1xTE Buffer.
16. Run on gel or use Spec to quantify DNA (use 5ul DNA/750ul 1x TE for OD
[260/280]. Store aliquots at either –80° C or -20°
To find amount of DNA OD260 = 1/50 ug/ul for d.s. DNA
SOLUTIONS
TE Buffer
10 mM Tris-HCl (pH 7.6)
1 mM EDTA (pH 8.0)
2x CTAB Isolation Buffer
100mM Tris-HCl (pH 8)
1.4 M NaCl
20mM EDTA
2% polyvinylpyrolidone (40,000 MW)
0.2% 2-mercaptoethanol  ADD ONLY IMMIDATELY PRIOR TO USE