Silica Clean-up of DNA

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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:

• 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
• 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

compiled by Chad Rappleye

Aroian Lab Protocols