Silica Clean-up of DNA

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