isolation of plant mitochondrial dna-protocol/buffer/solution composition??

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sidharth
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isolation of plant mitochondrial dna-protocol/buffer/solution composition??

can anyone please mail me the protocol and buffer/s composition for mitochondrial dna isolation..

cfish
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MITOCHONDRIAL DNA ISOLATION

MITOCHONDRIAL DNA ISOLATION

-Grind in mortar and pestle or Waring blender with 5-7 volumes buffer A per g tissue. Use MCE at 350 l/L, and if necessary, with 5 ml 1 M DIECA/L.
-Squeeze through cheesecloth, two layers of Miracloth.
-10 min at 1000 g
-Decant supernatant and centrifuge 10 min at 15,900 g.
-Resuspend each pellet in a few drops of buffer G with paint brush; combine; bring to about 10 ml/50 g, 15 ml/75 g.
-10 min at 1000 g; pour off most; swirl pellet to remove fluffy layer; combine.
-Bring supernatant to 10 mM MgCl2 (100 l 1M/10 ml). Bring to 20 g DNase/ml (100 l 2mg/ml/10 ml).
-60 min. 4 C.
-Underlay shelf buffer, 20 ml/10-15 ml; always use 20 ml or more.
-20 min at 12000 g.
-Resuspend in small volume shelf buffer with brush; bring to about 10 ml/50-100 g.
-10 min at 15900 g.
-Resuspend pellets in NN (lysis) buffer (4-5 ml/50-75 g).
-Add SDS to 0.5% (250 l of 10%/5 ml NN). Swirl thoroughly.
-Add proteinase K to 100 g/ml (25 l of 20 mg/ml/5 ml NN). Swirl gently.
-60 min. 37 C.
-Add equal volume of 3:1 water-saturated phenol, chloroform-isoamyl alcohol mixture. Emulsify ca. 5 min.
-10 min at 7000 g.
-Collect supernatant; repeat 17 and 18: 3 total extractions.
-Final supernatant; add 0.1 volume 8 M Ammonium acetate; then add 2 volumes of absolute ethanol.
-60 min, -80 C; 10 min at 8000-9000 g; drain; add equal volume 70% ethanol; let sit 10 min; 10 min at 8000-9000 g; drain dry. Vacuum dry pellet, 30 min. Two small corex tubes are better than one 30 ml Corex.
-Add 100-500 l 0.1X NTE, 10 l RNase mixture. Typically use 500 l per 50 g tissue.
-Hydrate 30 min., 37 C.

Link: http://wheat.pw.usda.gov/~lazo/methods/lazo/dnaplmit.html

sidharth
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cfish wrote:MITOCHONDRIAL DNA

cfish wrote:

MITOCHONDRIAL DNA ISOLATION

cfish,there is no mention of the composition of buffers a,g etc.

Tony Rook
Tony Rook's picture
sidarth:

sidarth:

Here is good reference for isolation of plant mitochondrial DNA...

Albert J. Wilson and Prem S. Chourey. A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources. Plant Cell Reports. Volume 3, Number 6 / December, 1984. 237-239

Abstract
A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can be performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA.

Weblink:

A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources

Fana
Fana's picture
i would like to know how

i would like to know how significant is the use of miracloth and cheesecloth in isolating mitochondrial DNA and what is the size range of plant mitchondrial DNA (ie in kbp)

msvnathan
msvnathan's picture
hi

hi
you can find the procedure for isolation of plant mitochondrial DNA from this article also. http://www.springerlink.com/content/c6l142w5w5415667/