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.) |
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