Genomic DNA from E.coli without phenol or chloroform??

Michael D Kawalek kawalekm at watserv.ucr.edu
Fri Nov 4 14:09:25 EST 1994


Gabriel Dorado (bb1dopeg at lucano.uco.es) wrote:
: Anybody knows a reliable method to obtain genomic DNA from E coli
: without the need of hazardous substances like phenol or chloroform? The

PEG and NH4 acetate precipitations can be used as alternatives to organic
extractions.  After making the lysate with SDS and protease K, add 1/2 volume\
of 30% PEG8000 3M NaCl.  Chill on ice for 60 min, then pellet the DNA at 12kG.
Resupend the DNA pellet in TE then add 1/2 volume of 7.5 M NH4 acetate.  \
Chill on ice for 60 minutes to precipitate proteins and RNA.  Pellet as before.
Precipitate the DNA from solution with 2 vol of ethanol and pellet again.\
Wash the DNA thoroughly to get rid of the salt.  You may want to do one more
precipitation with Na acetate and ethanol to get rid of any residual SDS
This DNA will be clean enough to digest with restriction enzymes.



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