Mini DNA extraction from wheat leaves for PCR and Southern B

Procedure for 100 ~ 300 mg of tissue.

1. Collect fresh tissue, coleoptiles or small amount of leaf tissue.  Use about three inches of a leaf blade. Preferably younger tissue. Fold leaf and place in a 2 ml microfuge tube.

2. Grind in liquid nitrogen to a fine powder.(The way we do this is to grind with a Kontes pestle attached to a drill and grind directly in the microfuge tube, kind of speeds it up, the blue plastic reusable pestles fit down into the 2.0 ml tubes)

3. Add 500 μl of Cornell extraction buffer (prewarmed to 65 ℃).

4. Vortex till suspended.

5. Place in 65 ℃ water bath for 45 minutes. (mixing by vortexing every ten minutes or so.)

6. Cool slightly and add 500 μl 24:1 chloroform:isoamyl alcohol. Shake vigorously or vortex till homogeneous.

7. Centrifuge 10 minutes at room temperature. (If you centrifuge at 4 ℃, SDS may re-precipitate.)

8. Draw off 400 μl supernatant into a new 2 ml tube containing 1 ml of 100 % ethanol.

9. Allow your sample to sit undisturbed for at least 15 minutes then invert gently several times. (IF you mix vigorously at this stage you will trap lots of impurities in with your precipitated DNA.)

10. Spin briefly, 10 sec, and aspirate off supernatant.

11. Wash with 1 ml 70% ethanol till pellet is clear and white. (the tubes can be set on their sides on a shaker overnight, or forever, or just a quick wash if the pellet is clean. The wash can be repeated if the pellet is still dirty.)

12. Spin briefly, dump supe, and let dry.

13. Dissolve DNA in 50 μl H2O. After adding water, set tubes upright on shaker overnight to allow your DNA to be resuspended. This long treatment will also result in the remaining RNA being degraded.......(100-200 mg, might yield 200 ug of DNA).

14. Store at 4 ℃. (long term at -20 ℃)

For a wheat southern blot a decent signal can be achieved by restriction digesting 10 μl of the 50 μl prep. Digest the 10 μl in a 20 μl reaction for 5 hours or overnight and run the DNA on 0.8 % agarose gels using TBE.

No need for RNAse treatment.

Extraction buffer 100 mls.

500 mM NaCl                     12.5  mls   of      4 M NaCl

100 mM Tris-HCl, pH 8.0       10  mls            1 M Tris

50 mM EDTA                        10  mls            0.5 M EDTA

0.84% SDS                           8.4 mls             10% SDS

Buffer is brought up in 90 % of final volume, equilibrated to 65 C, and then 0.38 g Sodium Bisulfite/100 ml buffer is added, and the warm buffers pH is adjusted to 7.8 ~ 8.0 with NaOH.