DESCRIPTION AND MATERIALS

• purify DNA from any type of agarose;
• concentrate DNA (for changing buffer, desalting);
• remove proteins (after restriction enzyme treatment; dephosporylation with BAP or CIAP);
• remove unincorporated nucleotides, primer, primer-dimers and enzymes from a labeling reaction or PCR;
• remove an excess of linkers after ligation;
• remove residual phenol, chloroform, and ethidium bromide.
• purify plasmid DNA free of RNA from bacterial lysates.

MATERIALS

1. Silica Powder Suspension: 50% in water (Silica from Sigma S-5631, washed with water 3-4 times by centrifugation)
2. Guanidine HCl or Guanidine Thiocyanate solution: 6M .(or DNAzol from GIBCO-BRL)
3. Washing Buffer: 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 50 mM NaCl, 50% ethanol
4. Elution:  deionized water or TE buffer

The silica mix (pre-made up to 10 ml) and 6M Gu-HCl or 6M GuSCN solution can be stored in a refrigerator at +4^o C up to six months. The prepared wash buffer should be stored in the freezer at -20^o C or made up fresh.
If the silica powder suspension looses liquid, add sterile distilled water to a volume that is approximately equal to the volume of the solid.
 
 

PROCEDURES

• Recovering DNA from any type of agarose gel
• Remove unincorporated nucleotides, primer, primer-dimers and enzymes from a labeling reaction or PCR
• Recovering DNA from small amount of tissues for PCR
• concentrate DNA (for changing buffer, desalting); Method is same as for PCR without STEP 1
• remove proteins (after restriction enzyme treatment; dephosporylation with BAP or CIAP) Method is same as for PCR without STEP 1
• remove an excess of linkers after ligation. Method is same as for PCR without STEP 1
• remove residual phenol, or ethidium bromide. Method is same as for PCR without STEP 1
• purify plasmid DNA free of RNA from bacterial lysates.

TROUBLE SHOOTING
 
 

Recovering DNA from any type of agarose gel;

Step 1. Run the agarose gel to separate DNA fragments. Estimate the concentration of DNA by comparing its intensity with that of a DNA standard of a similar size and known concentration. Cut agarose gel band containing the desired DNA. (Minimize the time of UV exposure as much as it is possible.) Determine an approximate volume of gel slice by weight (1g equals approximately 1ml) and place the slice into a plastic tube.
Step 2. Add at least 3 volumes of 6M Gu-HCl or 6M GuSCN solution (the final concentration must be at least 4 M Gu-HCl). Incubate 10 minutes at 57^o C to dissolve agarose (mix occasionally to promote agarose solubilization). If the solubilization is incomplete add one more volume of 6M Gu-HCl and incubate 5 more minutes.
Step 3. Add the resuspended silica powder suspension. Up to 2.0 mg of DNA add 5 ml of silica powder suspension, above 2.0 mg of DNA add 2 ml of silica powder suspension per 1 mg of DNA and incubate for 5 minutes at 57^o C. (mix occasionally to keep silica resuspended in the solution, do not vortex)
Step 4. Spin (all centrifugation steps are at 10,000 rpm in a tabletop microcentrifuge) silica powder/DNA complex for 15 seconds to form a pellet . Remove the supernatant. The pelleted silica powder/DNA is resuspended in 500 m l of 6M Gu-HCl solution to dissolve any agarose that has not dissolved during step 2. Place suspension in a 57^o C water bath for one minute and pellet at 10,000 rpm for 15 seconds. Remove the supernatant.
Step 5. Add 500 m l of ice cold wash buffer to the pellet. Resuspend the pellet in the wash buffer by pipetting or vortexing the pellet (do not vortex high molecular weight DNA). Centrifuge for 15 seconds in the centrifuge and discard the supernatant. Repeat the wash procedure two more times. During each wash the pellet should be resuspended completely. After the supernatant from the last wash has been removed, spin the tube again and remove the remaining liquid with pipette tip. The silica should be relatively ethanol free otherwise the remaining ethanol may inhibit enzyme reactions (the silica can be airdryed or vacuum dried for a few minutes, however overdrying my lead to poor recovery).
Step 6. Resuspend the silica powder/ DNA with TE buffer or water by vortexing/or pipetting. Use at least twice the volume of the original silica mixture used. Incubate the tube at 57⁰ C for 5-10 minutes. Mix and centrifuge for 30 seconds and carefully remove the supernatant containing the eluted DNA and place in a new tube. You can increase the elution efficiency using a large volume and performing 2-3 elution cycles with the new portion of eluant. Typically yields are greater than 80% in the first elution. In order to remove small amounts of the silica powder present in the eluate, spin the tube again for 30seconds in a table-top centrifuge and transfer the supernatant into a new tube (small amount of silica do not interfere with any type of enzyme assays, we frequently use silica/DNA complex for PCR, if the amount of silica is less than 5 m l). Eluted DNA can be used immediately in enzymatic or other manipulations.
Elute DNA into water or TE buffer.
 
 

Isolation of picogram quantities of DNA from tissue sections

Alternatively if you try to use the same material for several PCR reaction the mixture can be divided into several tubes or the DNA can be eluted with water or TE buffer. Use at least twice the volume of the original silica mixture used. Incubate the tube at 57⁰ C for 5-10 minutes. Mix and centrifuge for 30 seconds and carefully remove the supernatant containing the eluted DNA and place in a new tube.

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