Silica Clean-up of DNA
Materials:
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
if gel slice, melt at 55 degrees for 5 minutes with occasional agitation
for aqueous solutions add 1/10 vol 3M NaOAc to ensure pH is less than 7
2.Bind DNA to silica
add appropriate volume of silica suspension (binds approx 200 ng of DNA / ul of suspension) and mix well
sediment silica matrix by centrifugation (10 seconds in 'nanofuge' or brief spin at full spend in microfuge)
wash silica 3 times with 500 ul wash solution
pellet silica once more to remove any residual wash solution
air dry to remove any residual EtOH
3.Elute DNA from silica
resuspend silica pellet in desired volume of 10 mM Tris 7.5 or TE or 1X restriction buffer for downstream digests
heat 5 minutes at 60 degrees
spin 2 min @ full speed in microcentrifuge
remove DNA -containing supernatant to clean tube
Notes & Misc:
1.Conveinient PCR clean up before digestion: 100 ul PCR reaction cleaned up with 50 ul silica and eluted from silica with 30 ul 1X restriction buffer
2.Procedure useful for cleaning DNA from PCR reactions, restriction digests, etc. as well as purifying from agarose gel slices
3.If 6 M NaI solution becomes yellow, it will still bind DNA if 1/200 volume 10% acetic acid is added to lower pH
4.Procedure also useful for concentrating DNA from dilute solutions
References:
1.An inexpensive alternative to glassmilk for DNA purification. Boyle, J.S. and Lew, A.M. (1995). Trends in Genetics 11(1):8
2.GeneClean Protocols. Bio101
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