DNAandRNAEXTRACTIONS

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 4M LiCl 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% SDS50mM 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.