Arabidopsis DNA miniprep

kunkel at GARNET.BERKELEY.EDU kunkel at GARNET.BERKELEY.EDU
Mon Jan 6 14:23:52 EST 1992






Plant DNA Mini-Prep 	

Modified By Doug Dahlbeck from T. Tai and S. Tanksley, 1991, Plant 
Mol. Biol. Rep. 8:297-303.  

This protocol works very well for Arabidopsis, and can be easily scaled
up for larger amounts of tissue.  

EXTRACTION BUFFER		
100 mM Tris-HCI pH 8	
50 mM EDTA pH 8		
500 mM NaCI			
1.25% SDS (w/v)			
8.3 mN NaOH			
0.38% Na bisulfite		

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 microl T5E, vortex 2 sec., into 65oC 5 min., vortex 2 sec 
(be sure pellet  resuspends).

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

9. Transfer into a new 1.5 ml tube, add 330 microl 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 microl T5E, vortex 2 sec., heat 65o C for 5 min., and vortex 2 
sec.

12. Add 10 microl 3 M NaOAc, mix, add 75 microl 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 microl 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.

	We have had great success using this procedure to prepare DNA from
large leaves of older Arabidopsis plants.  
	Good Luck - Barbara.Kunkel.





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