Purification of Genomic DNA by Ethanol Precipitation Materials Needed 100% Ethanol 70% Ethanol 3M Sodium Acetate Sterile Water

OR 10mM Tris pH 7. ! ". Precipitate the DNA 1. Add # times $our %&A sample 'olume o( 100% cold ethanol. )or e*ample+ i( $ou started ,ith 100 -l o( .%&A /0.enomic %&A1+ use #00 -2 o( 100% ethanol. Total 'olume should not e*ceed 1. m2. #. Add 1310 o( the ori.inal sample 'olume o( 3M &aOAc. A.ain i( ,e started ,ith 100 -l o( .%&A+ ,e4ll add 10 -l o( &aOAc. 3. Mi* 5$ 6uic7l$ in'ertin. se'eral times until ,ell com5ined. 8ou ma$ see a 9cloud$: or 9cotton 5all;li7e: precipitate 5e.in to (orm at this point. <. =ut the tu5e/s1 at ;#0> (or #0 minutes to (acilitate precipitation. Pellet the DNA . Spin (or 10 minutes at 13;1<7 rpm in a ta5letop centri(u.e. &ote ho, $our tu5es are oriented in the centri(u.e+ as this ,ill .i'e a clue as to precisel$ ,here to loo7 (or $our pellet. ?. Remo'e the supernatant li6uid (rom the tu5es 5$ pourin.+ pipettin.+ or 'acuumin. ,hile making sure not to disturb/lose the pellet. 8ou ma$ see a small ,hite or translucent pellet at this point. @( not+ assume it is ,here $ou e*pect it to 5e. Wash the Pellet 1. Add 00 -l o( 70% ethanol. 8ou ma$ ,ish to tr$ to dislod.e the pellet 5$ aimin. directl$ a5o'e the pellet+ and e*pellin. (orce(ull$. @n'ert se'eral times to thorou.hl$ ,ash. #. Spin (or minutes at 13;1<7 rpm. 3. A.ain+ remo'e the supernatant li6uid (rom the tu5es 5$ pourin.+ pipettin.+ or 'acuumin. ,hile making sure not to disturb/lose the pellet. <. Air;dr$ the pellets Aust until ethanol is no lon.er 'isi5le+ 5ut pellet doesn4t $et loo7 dr$ or crac7ed+ a5out ;10 minutes. O'er;dr$in. ,ill lead to di((icult$ in resuspendin. the %&A. Resuspend the DNA 1. Resuspend the %&A 5$ addin. an appropriate 'olume o( nuclease;(ree ,ater or 10mM Tris pH 7. ; ". and pipettin. up and do,n to mi*. @( $ou can dislod.e the pellet ,hile doin. this+ resuspension ,ill 5e (aster. Warmin. the %&A /37; B>1 can speed up resuspension.
Ho, much li6uid to use (or resuspendin. $our pellet depends on ho, much %&A $ou4re e*pectin. to .et out+ and ,hat concentration $ou4d li7e $our %&A stoc7 to 5e. We .enerall$ li7e %&A stoc7s 5et,een 100 and #00 n.3-l. 8ou can assume a5out ?0% o( $our startin. material has made it to this point+ and use that num5er to decide on a li6uid 'olume. )ull$ resuspended %&A should .i'e consistent spec readin.s throu.hout.