Summary Plant DNA extractions

Todd Richmond todd at GENETICS.WISC.EDU
Thu Feb 13 10:03:08 EST 1992


A while ago I posted a couple of requests for
preps for nuclear, mitochondrial and chloroplast
DNA.  Here's a summary of some of the references
I managed to dig up and some protocols sent to
me by various individuals on the net.  Thanks 
to everyone who responded.  I haven't tried any of 
these preps yet so if you have and have any comments
to add, please share them.  If you have something
better that you use in your lab, feel free to 
post it or email it to me.

Thanks,

Todd

List of individual references:

Review:
Methods in Enzymology Volume XXXI Biomembranes Part A
Sidney Fleischer and Lester Packer, eds.
1974
     *has a number of different protocols for different
      organelles and a discussion of various factors
      affecting the yield and intactness of the prep

***************************************************************
Isolation of nuclei:
Luthe, D.S. and Quatrano, R.S., Transcription in Isolated 
Wheat Nuclei, Plant Physiol. (1980) 65, 305-308.
   *three methods given: a crude prep, one utilizing a 
    Percoll gradient, and one utilizing a sucrose gradient

Slater, R.J., Venis, M.A., and Grierson, D., Characterization
of Ribonucleic Acid Synthesis by Nuclei Isolated from Zea
Mays, Planta 144, 89-93.  (1978)
    *gives intact nuclei but probably contaminated with 
     chloroplasts as well?

Wilson, P.S. and Bennett, J., 1976, Transcription in 
Nuclei Isolated from a Higher Plant, Biochemical
Society Transactions 4:812-813.
    *references Hamilton et al., 1972 below.  Notes 
     that the initial nuclear pellet was heavily 
     contaminated with chloroplasts but these were
     removed by repeated washing in hyperosmotic
     sucrose solution containing Triton X-100 and
     Mg2+ ions.

Hamilton, R.H., Kunsch, U. and Temperli, A., (1972)
Anal. Biochem. 49:48-57.
*************************************************************
Chloroplast References:

Mulligan, B.G., Purification of Chloroplast DNA using
Hexadecyltrimethylammonium Bromide (CTAB), (1989),  
Plant Molecular Biology Reporter 7(2):144-149.

Dally, A.D. and Second, G., Chloroplast DNA isolation
from Higher Plants:  An improved non-aqueous method,
(1989), Plant Molecular Biology Reporter 7(2):135-143.

Mourad, G. and Polacco, M.L., Mini-preparation of 
Highly Purified Chloroplast DNA from Maize, (1989),
Plant Molecular Biology Reporter 7(1):78-84.

*************************************************************
Mitochondria Prep
ref:  Sparks, R.B., Jr. and Dale, R.M.K., Characterization of
3H-Labeled Supercoiled Mitochondrial DNA from Tobacco
Suspension Culture Cells, Molec. Gen. Genet. (1980) 180:351-355.

Mitochondrial Prep from Suspension cells
1.  Begin with 300-600 g of cells (can be scaled 
down to 100 g)

2.  Filter cells through miracloth (this step
optional if cells not clumpy)

3.  Weigh cells

4.  Dilute cells 1:1 with RD-MP buffer #1

5.  Break cells by passing through french press
at 3,000 psi

6.  Collect cells in chilled flask in ice

7.  Filter solution through two layers miracloth

8.  Centrifuge 1,500 g at 4 C

9.  Discard pellet

10.  Centrifuge 1,500 g at 4 C for 15 minutes

11.  Discard supernatant and resuspend in RD-MP buffer
#1 w/o PVP, adjust to 10 mM MgCl2, and then treat with 
DNase (50 ug/ml) for 1 hour at 20 C.

12.  Adjust sample to 20 mM EDTA and centrifuge again
at 15,000 g for 15 min

13.  Resuspend pellet by homogenization in a glass
homogenizer in 36 ml of RD-MP buffer #2.

14.  Layer on discontinuous sucrose gradient made in
the same buffer.
    Gradient:   6 ml 1.6 M sucrose
                8 ml 1.4 M sucrose
                8 ml 1.2 M sucrose
                4 ml 0.9 M sucrose
                4 ml 0.6 M sucrose
                6 ml sample

15.  Centrifuge 1 hr in a SW27 rotor at 25,000 rpm at 4 C.

16.  The mitochondria will band at the 1.4/1.2 interface.
Remove them with a syringe through the side of the tube.

17.  Dilute 1:3 with RD-MP Buffer #2

To obtain DNA:
18.  Centrifuge mit. solution for 20 min. at 15,000 g.

19.  Resuspend in 7.5 ml of RD-MP Buffer III made 
0.5% in Sarkosyl.

20.  Immediately layer on a CsCl-EtBr gradient and
centrifuge for 40 hours in a Ti50 rotor at 38,000 rpm.

21.  Open-circular/linear and supercoil bands can be
seen by long wave illumination and removed with a 
syringe through the side of the tube.

22.  Extract EtBr with CsCl saturated isopropanol.

23.  Dialyze the DNA sample against two changes of
TEN made 0.3 M in sodium acetate and then EtOH ppt.

24.  Resuspend the ppt'd DNA in water, TEN, or TE
and store at 4 C.

Buffers:

RD-MP Buffer #1
0.3 M mannitol
0.05 M Tris-HCL
3 mM EDTA
before use add:
1 mM 2-mercaptoethanol
0.1% BSA
1% Polyvinylpyrrolidone, PVP-40
 pH to 8.0

RD-MP Buffer #2
0.3 M sucrose
0.05 M Tris-HCL
0.02 M EDTA
before use add:
0.1% BSA
 pH to 8.0  

RD-MP Buffer #3
0.05 M Tris-HCL
0.02 M EDTA
 pH to 8.0

TEN
10 mM Tris
50 mM NaCl
5 mM EDTA
 pH to 8.0

**********************************************************
Other protocols gathered from the net:



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