Dried Plant DNA Prep

MOUNTLAB at CCIT.ARIZONA.EDU MOUNTLAB at CCIT.ARIZONA.EDU
Wed Jun 3 12:30:00 EST 1992


			Plant DNA Isolation
                            Adapted from Focus 12(1): 13-15 (1990) by GH
				


  1.  Preheat isolation buffer to 60 C.
  2.  Grind fresh tissue in clean mortar:pestle w/ liq N2 (tissue:buffer = 0.5 g/ 5 ml).
  3.  Immediately add preheated isolation buffer and place in 60 C water bath.
  4.  Incubate at 60 C for 30 min.
  5.  Extract once w/ an equal volume of chloroform:isoamyl-OH (24:1).
  6.  Spin in GLC-1 for 5 min. on RmaxS.
  7.  Transfer supernant to a separate tube.
  8.  Add E 2/3 volume -20 C isopropanol to supernant (7).
  9.  Gently rock tube back and forth until DNA precipitates.
10.  Pellet or spool DNA - spin E 2 min GLC-1.
11.  Transfer pellet to 1.5 ml eppendorf tube filled w/ 0.75 ml 60 C isolation buffer.
12.  Incubate at 60 C for 30 min.
13.  Extract once w/ an equal volume of chloroform:isoamyl-OH (24:1).
14.  Spin in Microfuge for 5 min. on RmaxS.
15.  Transfer supernant to a separate tube.
16.  Add E 2/3 volume -20 C isopropanol to supernant (15).
17.  Gently rock tube back and forth until DNA precipitates.
18.  Pellet or spool DNA - spin E 2 min in Microfuge 40% power.
19.  Pour liquid off and rinse with r.t. 70% EtOH (twice).

Dry thoroughly in Speed-vac (15-20 min.).
Resuspend in TE - store at 4 C.



Isolation buffer:			100 ml

CTAB	(2% solution w/v)		    2.00 g  
NaCl   	(1.4 M)			    7.68 g
b-mercaptoethanol (0.2% v/v)		   200 ul
EDTA (20 mM)			     4 ml    of a 0.5 M
Tris-HCl, pH 8.0 (100 mM)		   10 ml    of a 1.0 M

Add ddH20  for a final volume of         100 ml
Store at room temp					


Our lab has used this protocol for various different species and have had great success with it.  Good Luck.
Greg Harlow, David Mount's Lab.



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