Silica Suspension: add 2 g of silica to 15 ml of H2O wash 3x by centifugation at 2000 x g for 2 min estimate vol of silica and resuspend in 2 vol H2O Silica Wash Solution: 50 mM NaCl,10 mM Tris 7.5,2.5 mM EDTA,50% Ethanol 6 M NaI note: light sensitive Procedure: 1.Add 5 volumes of 6M NaI to DNA solution or agarose gel slice oif gel slice,melt at 55 degrees for 5 minutes with occasional agitation ofor aqueous solutions add 1/10 vol 3M NaOAc to ensure pH is less than 7 2.Bind DNA to silica oadd appropriate volume of silica suspension (binds approx 200 ng of DNA / μl of suspension)and mix well osediment silica matrix by centrifugation (10 seconds in 'nanofuge' or brief spin at full spend in microfuge) owash silica 3 times with 500μl wash solution opellet silica once more to remove any residual wash solution oair dry to remove any residual EtOH 3.Elute DNA from silica oresuspend silica pellet in desired volume of 10 mM Tris 7.5 or TE or 1X restriction buffer for downstream digests oheat 5 minutes at 60 degrees ospin 2 min @ full speed in microcentrifuge oremove DNA-containing supernatant to clean tube Notes & Misc: •Conveinient PCR clean up before digestion: 100μl PCR reaction cleaned up with 50μl silica and eluted from silica with 30μl 1X restriction buffer •Procedure useful for cleaning DNA from PCR reactions,restriction digests,etc.as well as purifying from agarose gel slices •If 6 M NaI solution becomes yellow,it will still bind DNA if 1/200 volume 10% acetic acid is added to lower pH •Procedure also useful for concentrating DNA from dilute solutions References: •An inexpensive alternative to glassmilk for DNA purification.Boyle,J.S.and Lew,A.M.(1995).Trends in Genetics 11(1):8 •GeneClean Protocols.Bio101 |
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