Alex Ma: New page: ==Overview== DNA Cleanup protocol adapted from http://www-ufk.med.uni-rostock.de/lablinks/protocols/e_protocols/phenolch.htm removes protein debris and resuspends DNA in appropriate final...

Alex Ma — Mon, 02 Apr 2012 15:43:04 GMT

New page: ==Overview== DNA Cleanup protocol adapted from http://www-ufk.med.uni-rostock.de/lablinks/protocols/e_protocols/phenolch.htm removes protein debris and resuspends DNA in appropriate final...

New page

==Overview==

DNA Cleanup protocol adapted from http://www-ufk.med.uni-rostock.de/lablinks/protocols/e_protocols/phenolch.htm removes protein debris and resuspends DNA in appropriate final buffer.

==Materials==

*1.5 ml microcentrifuge tubes (eppies)
*DNA of interest
*Phenol
*Chloroform
*Ethanol
*3 M sodium acetate
*10mM tris pH 7.5

==Procedure==

===Phenol Step (removes protein)===

#Pipette 500 µl DNA suspension into eppendorf tube (can vary volume). Add an equal volume of phenol (tris-saturated Phenol-Chloroform-Isoamyethanol).
#Vortex, then centrifuge for 2 min @ 12000 rpm 4°C.
#Transfer supernatant into new tube, avoiding drawing the interlayer or organic phase.

===Chloroform Step (removes phenol)===

#Add an equal volume of chloroform to the supernatant from previous step.
#Vortex, then centrifuge for 2 min @ 12000 rpm 4°C.
#Transfer supernatant into new tube, avoiding drawing the interlayer or organic phase.

===100% Ethanol Step (precipitates DNA)===

#Add 0.1 volume 3 M sodium acetate.
#Add 2.5 volumes 100% ethanol.
#Vortex and precipitate at -20°C overnight. Can also precipitate at -80°C for 1 hr or dry ice for 15 min, but precipitation won't be as complete as -20°C overnight.
#Centrifuge for 20 min at 12000 rpm 4°C.
#Carefully pour out or aspirate supernatant, taking care not to lose DNA pellet.

===70% Ethanol Step (washes out salt)===

#Carefully add 1 ml cold 70% ethanol. DO NOT VORTEX.
#Centrifuge for 10 min at 12000 rpm 4°C.
#Carefully pour out or aspirate supernatant, taking care not to lose DNA pellet.
#Air dry for 10 min at RT, but don't overdry (DNA becomes hard to dissolve).
#Dissolve in 10 mM tris pH 7.5.
#*TE-Buffer will work also, but EDTA present may inhibit downstream enzymatic reactions.
#*dH2O can be used but must be frozen immediately at -20°C, since unbuffered DNA undergoes degradation.

[[Category:ShreffLab]]

[[Category:Protocol]]

[[Category:DNA]]

Shreffler:DNA Cleanup - Revision history

Alex Ma: New page: ==Overview== DNA Cleanup protocol adapted from http://www-ufk.med.uni-rostock.de/lablinks/protocols/e_protocols/phenolch.htm removes protein debris and resuspends DNA in appropriate final...