(none)

andrew2 at GARNET.BERKELEY.EDU andrew2 at GARNET.BERKELEY.EDU
Tue Jun 2 13:58:25 EST 1992


Dear Chuck et al.:

We posted this protocol a few months back - it is a modification of Tai &
Tanksley's procedure for DNA from dried material.  Not necessarily the
"best" prep, but it works fine.  It was developed by Tom Tai and Doug
Dahlbeck.







Plant DNA Mini-Prep
(modified from T. Tai and S. Tanksley, 1991, Plant Mol. Biol. Rep. 
8:297-303)

Solutions:

EXTRACTION BUFFER	for 10 ml			for 600 ml
100 mM Tris-HCI pH 8	1 ml  1M			60 ml 1 M
  50 mM EDTA pH 8	1 ml 0.5 M			60 ml 0.5M
500 mM NaCI	1 ml 5 M			60 ml 5 M
1.25% SDS (w/v)	1.25 ml 10%			75 ml 10 %
8.3 mN NaOH	83 ul 1N (8.3 of 10 N)	0.5 ml 10 N
0.38% Na bisulfite	38 mg				2.28 g

T5E
50 mM Tris pH 8
10 mM EDTA pH 8

5 M Potassium acetate

PROTOCOL:

1. Collect leaf tissue (up to 0.15 g) in eppendorf tube.  Freeze by 
dipping in liquid nitrogen to fill tube.  Process in sets of about six 
tubes: fill all tubes with liquid nitrogen, grind them all and proceed 
to step 2 before starting another set.  Alternatively, can allow liquid 
nitrogen to evaporate and then store samples at -70o for later use.

	Note:  Leaf tissue can also be dried overnight in a 45oC forced 
air oven, stored dry at room temperature, and then powdered prior 
to step 2.

2. Add 0.7 mls of extraction buffer that is preheated to 65o C and 
mix thoroughly using a toothpick or pipet tip.  Put in 65o C water 
bath for 10 min.

3. Add 0.22 mls 5 M Potassium Acetate, mix well and put on ice for 
20 to 40 min.

4. Pellet at 4o C for 3 min.

5. Filter supernatant.  Pour into blue tip that has a small kimwipe 
"filter", force liquid into another eppendorf tube using pipetman.  
Add 0.7 vol. (0.4 ml?) of IPA and mix well.

6  Pellet at 4o C for 3 min., pour out supernatant, and rinse pellet 
twice with 70% ETOH.  Drain (on test tube rack or paper towel) for 1 
min.

7. Add 300 ul ml T5E, vortex 2 sec., into 65oC 5 min., vortex 2 sec (be 
sure pellet  resuspends).

8. Add 150 ul 7.4 M NH4OAc, vortex 2 sec. and pellet 3 min.

9. Transfer into a new 1.5 ml tube, add 330 ul IPA and mix well.

10. Pellet 3 min., pour out supnt., rinse pellet twice with 70 % ETOH, 
and drain inverted for 2 min.

11. Add 100 ul T5E, vortex 2 sec., heat 65o C for 5 min., and vortex 2 
sec.

12. Add 10 ul 3 M NaOAc, mix, add 75 ul IPA, and mix well.

13. Pellet 3 min., pour out supnt., rinse pellet twice with 70 % ETOH, 
and drain inverted for 2 min.

14. Dry pellet in speedvac for 15 min., add 25 ul of TE and let pellet 
resuspend overnight at 4o C.

15. Vortex 2 sec., heat 65o C 5 min and vortex 2 sec.  Store at 4o.


Storing at -20o is better idea for longer periods.
Scale-ups of this (for 50 ml tubes, etc.) also work fine.




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