prepare DNA from flowering plant Azalea (rhododendron, Ericaceae)

dilip dilip at zeus.tamu.edu
Tue Aug 29 12:44:17 EST 1995


NANCY:
I AM WORKING WITH DNA FROM PINE TISSUES WHICH HAS BEEN A PROBLEM TO 
ISOLATE SOME TIME BACK. I USED THE PROTOCOL DEVELOPED BY DOYLE AND 
DOYLE, FOCUS 12(1),13-15, WHERE THEY USED CTAB - HEXADECYLTRIMETHYL 
AMMONIUM BROMIDE (2%), 1.4 M NaCl, 0.2% beta MERCAPTOETHANOL, 20 mM 
EDTA, 100mM TRIS-HCl and PVP 2% TO ISOLATE DNA FROM PLANT TISSUES. USE 5 
- 7 MLS OF BUFFER TO EACH GRAM OF TISSUE GROUND TO POWDER IN LIQUID 
NITROGEN. INCUBATE THE TISSUE IN SOLUTION AT 55 C FOR 30 MIN. AND 
EXTRACT ONCE WITH CHLOROFOAM AND CENTRIFUGE TO REMOVE THE UPPER AQOUES 
SOLUTION THAT CONTAIN THE DNA AND RNA. PERCIPITATE THE NUCLEIC ACID WITH 
ISOPROPANOL OR ETOH. SPOOL THE DNA IMMEDIATELY OR AFTER ABOUT 5 MINUTES 
ON ICE. LONGER THE TIME YOU KEEP THE MORE THE OTHER THINGS CONTAMINATE 
THE DNA. INCREASING THE NaCl TO 2.0 mM IN THE EXTARCTION BUFFER MAY HELP 
TO BRING DOWN THE CARBOHYDRATE CONTAMINATION DOWN (THERE WAS A REPORT ON 
THIS). TRY IT AND LET ME KNOW.
DILIP DIAS
DILIP at ZEUS.TAMU.EDU
DEPT. OF FOREST SCIENCE
TEXAS A & M UNIVERSITY





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