DNA and RNA EXTRACTIONS

A protocol / method / schedule /procedure for extraction / isolation of both DNA and RNA from the same material typically plant leaf / leaves

(See also

1)Take one medium sized leaf or half a large leaf (5 to 20 cm^2),weigh and freeze in liquid nitrogen.

2)Grind the tissue in a bleached and baked pestle and mortar with liquid nitrogen.

3)Transfer the powder produced to a l5ml Falcon blue cap tube.Add 2 ml of homogenization buffer (4 ml per g)and disperse the tissue in it.

4)Leave for 10 min at room temperature.

5)Add 2 ml of phenol/chloroform and vortex.

OR,

1)Put a small or medium sized leaf into a 4" x 6" 500 guage (thick!)plastic bag.

2)Add 2-3 ml homogenization buffer (you may need more for large leaves)to the bag.

3)Grind the leaf inside the bag using the top end of a 50 ml corex tube.

4)Pour the homogenate immediately into a 15 ml Falcon blue cap tube containing equal volume of phenol/chloroform then vortex.

5)Goto step 6

6)Spin for 10 min at 2,500 rpm in a bench centrifuge.

7)Transfer 0.7 ml of the aqueous phase to an Eppendorf tube,add 0.7 ml phenol/chloroform,vortex and spin for 5 min.

8)Transfer 0.6 ml of the aqueous phase to a fresh Eppendorf tube,add 0.6 ml phenol/chloroform,vortex and spin for 5 min.

9)Transfer 0.5 ml of the aqueous phase to a fresh Eppendorf tube,add 0.5 ml chloroform/isoamyl alcohol,vortex and spin for 5 min.

10)Add 4mliCl to the final conc.of 2M and leave for several hrs at 4℃ (better o/n).Spin for 10-15 min.RNA should be in a pellet.Remove supernatant and precipitate the DNA with ethanol.

11)Treat RNA fraction with DNase RNase-free,and the DNA fraction with RNase-free of DNase.

Notes:

Homogenization buffer:

0.1 M NaCl 2% SDS

50mM Tris-HCl pH 9.0 10 mM EDTA

(ideally DEPC treated water and everything else RNase free)

then add 0.1 mg/ml proteinase K { added fresh }

Pat Heslop-Harrison University of Leicester December 2003.

(I'm afraid I did not note the source of this protocol.)