Nucleic Acid Purification
There are many methods available for purifying nucleic acids. The choice of method depends on the type, source, size and amount, and quality required for the procedure in which the nucleic acid is to be used. The advantages and disadvantages of each purification method are presented in Table 1 (See notes/conditions & reference protocols).

Phenol Extraction (Sambrook)
Phenol dissociates proteins from DNA. Chloroform denatures the protein and lipids and helps maintain the separation of the organic and aqueous phase. It also makes the DNA less soluble in the phenol, thus reducing losses to the organic phase. Isoamyl alcohol is often added to prevent foaming. At pH 7-8, the DNA partitions to the aqueous phase while the protein form an opaque layer at the between the phases. By adjusting the phenol to pH 5-6, DNA will be retained in the organic phase while RNA will be retained in the aqueous phase. Lower sodium ion concentrations favor improved yields of RNA and oligonucleotides with this method.

Guanidinium isothiocyanate (Chomczynski)
Guanidinium isothiocyanate, one of the most powerful protein denaturants, have been very effective in RNA purification methods due to the ability of the reagent to suppress Rnase activity. Guanidinium isothiocyanate used in combination with phenol/chloroform extraction and alcohol precipitation provides quality intact RNA.

Alcohol Precipitation (Sambrook, Methods Enzymology 152)
Adding monovalent cations (K+, Na+, NH₄+) to nucleic acid solutions causes salts to form with the negatively charged nucleic acids. Adding alcohol (ethanol or isopropanol) causes the nucleic acids to precipitate. Although less alcohol is required with isopropanol methods, the salts are not as soluble in isopropanol and may co-precipitate with the nucleic acids. Precipitations are typically performed at -20°C. However, more recently, it was found that ammonium acetate precipitation may be carried out at room temperature. Ammonium acetate is frequently used to reduce the precipitation of dNTPs. Lithium chloride should be avoided when the RNA is to be used for cell-free translation or reverse transcription. Sodium chloride is desired if the nucleic acid sample contains SDS because the detergent will precipitate with other systems.

RNA
RNA may be precipitated with ethanol (or isopropanol) after adjustment with 0.2 M Na+ or K+ (or 0.5 M NH₄+) cations. The RNA is precipitated by adding 2.5 volumes of ethanol (or 1 volume of isopropanol), mixing and freezing (> -20°C) for at least 15 minutes. Precipitation works best at RNA concentrations of at least 10 µg/ml. Alternatively, 4 M lithium chloride (LiCl) in 10 mM Tris, 10 mM EDTA, pH 7.4 may be added in equal volume to RNA and frozen (> 20°C) for several hours to overnight. Pellets are centrifuged and rinsed with 70% ethanol to remove trace LiCl. This method is much less likely to have carbohydrate, protein or DNA contamination. This precipitation works best at RNA concentrations of at least 200 µg/ml. Lithium chloride precipitation may be used for in vitro purification where lysis and high protein content is not an issue.

DNA
DNA may be precipitated with ethanol (or isopropanol) after adjustment with 0.2 M sodium chloride, 0.3 M sodium acetate, 0.8 M lithium chloride or 2-2.5 M ammonium acetate. Sodium acetate
(0.3 M; pH 5.2) is used for most DNA and RNA precipitation.

DEAE (Ion Exchange)
DEAE can be purchased as resin or paper. The negative phosphate backbone of DNA binds to anionic DEAE in low ionic strength (low salt) buffer such as 10 mM Tris pH 7.5, 1 mM EDTA and 0.2 M NaCl. DNA is eluted by increasing the salt concentration (to ~2 M) in the elution buffer. Nucleic acids may be collected on DEAE paper by inserting a section of DEAE paper below the band of interest on an agarose gel. Electrical current is applied to allow the nucleic acid to be collected on paper. High salt must be removed by dialysis or desalting.

Size Exclusion Chromatography (SEC)
Nucleic acids are applied to porous beads that exclude the nucleic acid while trapping smaller salts, nucleotides and derivatization reagents in the internal pores of the beads. The SEC columns are often packed as small centrifugal devices.

Cesium Chloride
Separation of plasmid from genomic DNA is based on the differential amounts of ethidium bromide incorporation and the buoyancy exhibited in the cesium chloride concentration gradient.

Nucleic Acid Extraction from Agarose
Gel purification of nucleic acids involves separating the mixture out on a gel, removing the band of interest with a razor blade, and then extracting the nucleic acid from the agarose. Nucleic acids may be extracted from agarose by electroelution using dialysis membrane or membrane devices; ion exchange by electrophoresis onto anion paper; liquification by phenol or chaotrophic salts followed by phenol or silica purification methods, respectively or purification by centrifugation with homemade or commercial devices.

Extraction from Agarose
A purification device can be created by cutting the bottom off a 0.5 ml microtube and then placing sterile glass wool or pillow stuffing in the tube. Place a 1.5 ml catch tube over the 0.5 ml tube. The gel slice can be placed on top of the pillow stuffing, frozen at -20°C for 30 minutes and then spun for 10 minutes at 10,000 x g.

Silica
In the presence of high chaotrophic salts, nucleic acids will bind to silica particles with great affinity. The chaotrophic salts denature the proteins and break down the polymeric structure of agarose. Denatured proteins and agarose monomers can be selectively washed from the silica with ethanol containing wash buffers. The pH of the binding and wash solutions and the concentration of the ethanol are critical conditions for isolating different size and strand nucleic acids. The nucleic acid can then be eluted with low-salt buffer (TE or water). Using a warmed elution buffer (50-60°C) may improve nucleic acid recovery.

Notes/Conditions

Phenol:Chloroform:Isoamyl Alcohol (PCIAA) Extraction

1. Make PCIAA by adding 25 parts of phenol (Tris-HCl saturated pH >7.4) + 24 parts of chloroform + 1 part isoamyl alcohol. Vortex thoroughly. Add the top aqueous layer of the phenol solution for storage up to 3 weeks at 4°C protected from light.
2. Add equal volume of PCIAA to DNA mixture.
3. Vortex thoroughly. Centrifuge at 12,000 x g for 20 minutes at room temperature.
4. Carefully remove top layer and place in a clean tube. Discard bottom phase.
5. Repeat steps 3 - 5 then proceed to ethanol precipitation.

1. Add 0.25X volume of 8 M ammonium acetate and 3X volume of 95% ethanol. Vortex thoroughly and centrifuge for 12,000 x g for 30 minutes. Do not freeze.
2. Remove supernatant. Be careful not to disturb pellet.
3. Overlay pellet with 80% ethanol and centrifuge at 12,000 x g for 30 minutes.
4. Remove supernatant. Be careful not to disturb pellet.
5. Invert the tube and allow to air dry.
6. Re-suspend the pellet in nuclease free water (10-50 µl depending on DNA quantity).

Total DNA Extraction From Bacteria (Maloy)

1. Re-suspend the pellet from 1.5 ml culture in 567 µl nuclease free water by repeated pipetting. Add 30 µl of 10% SDS and 3 µl of 20 mg/ml proteinase K, mix by inverting, and incubate 1 hour at 37°C.
2. Add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and thoroughly mix by inverting until an emulsion is formed.
3. Centrifuge 12,000 x g for 5 minutes at room temperature.
4. Transfer the top aqueous phase to a new tube. Leave a small amount of supernatant behind so that the interphase is not disturbed.
5. Add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Mix by inverting and centrifuge for 10 minutes at 12,000 x g.
6. Transfer the top phase to a new tube leaving a small amount of supernatant so that the interphase is not disturbed.
7. Add 0.6 volumes of isopropanol and invert gently until the DNA precipitates.
8. Spin for 10 minutes. Pour off supernatant and add 1 ml of ice-cold 70% ethanol. Spin for 5 minutes at 10,000 x g.
9. Decant the supernatant. Dry the DNA pellet in a speed vacuum for 10 minutes.
10. Re-suspend the pellet in nuclease-free water or TE. Check A260/A280 for purity.

Total DNA Extraction from Tissue (Laird)

1. Make lysis buffer: 5 ml of 100 mM Tris-HCl, pH 8.5 + 0.5 M EDTA + 1 ml 10% SDS + 2 ml 5 M NaCl + 0.25 ml 20 mg/ml Protease K + QS with 50 ml nuclease-free water.
2. Add lysis buffer to tissue (could be varying amounts, typically 5X volume).
3. Agitate for 2-3 hours at 55°C.
4. Add equal volume of isopropanol and mix gently until the DNA precipitates.
5. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end).
6. Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sececonds.
7. Re-suspend DNA in 100-200 µl nuclease free water. (This may take several hours).

RNA Purification
*Commercial stabilizer solutions can be added to the tissue or cells to protect the RNA from damage prior to purification.

Lithium Chloride Precipitation

1. Add 0.1X volume of 8 M LiCl.
2. Chill the tube at -20°C for 30 minutes.
3. Centrifuge tube for 15 minutes at 10,000 x g at 4°C.
4. Remove supernatant. Be careful not to disturb pellet.
5. Overlay pellet with ice-cold 80% ethanol and centrifuge for 12,000 x g for 30 minutes. Volume should be equal to that in part A.
6. Remove supernatant. Be careful not to disturb pellet.
7. Invert the tube and allow to air dry.
8. Re-suspend the pellet in nuclease-free water (50-10 µl depending on RNA quantity).
9. If removal of residual chloride ions is necessary, dialyze RNA sample with Slide-A-Lyzer MINI Dialysis Unit to remove chloride. Chloride ions will inhibit reverse transcriptase.

Guanidinium Thiocynate Total RNA Extraction (Chomczynski)

1. Prepare denaturing solution (4 M guanidinium thiocyanate,
   25 mM sodium acetate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol): Dissolve 50 g of guanidinium thiocynate in 60 ml of sterile nuclease-free water. Add 1 ml of 2.5 M sodium acetate pH 7.0 +1 ml of 10% sarcosyl at 65°C. This solution is stable for at least 3 months at room temperature. Add 70 µl of 2-mercaptoethanol per 10 ml of denaturing stock solution.
2. Add 1 g of a tissue sample or 5 x 10⁷ pelleted culture cells to 10 ml of denaturing solution and homogenize in a glass-PTFE homogenizer.
3. Add 1 ml of 2 M sodium acetate, pH 4 + 10 ml of phenol (water saturated) + 2 ml chloroform:isoamyl alcohol (49:1) and vortex for 10 minutes and chill on ice for 30 minutes.
4. Centrifuge for 20 minutes at 12,000 x g at 4°C.
5. Transfer top phase to a clean tube. Do not disturb interphase.
6. Add 10 ml of ice-cold isopropanol. Incubate at -20°C for 2 hours.
7. Centrifuge for 20 minutes at 10,000 x g at 4°C.
8. Remove supernatant, be careful not to disturb pellet.
9. Re-suspend pellet in 1.5 ml of denaturing solution.
10. Add 1.5 ml of isopropanol and chill at -20°C for 1 hour.
11. Remove supernatant, be careful not to disturb pellet.
12. Overlay pellet with ice-cold 75% ethanol and centrifuge for 12,000 x g for 30 minutes.
13. Remove supernatant. Be careful not to disturb pellet.
14. Invert the tube and allow to air dry.
15. Re-suspend pellet in sterile nuclease-free water (50-100 µl depending on RNA quantity and pellet size)

Tips

• Use good laboratory practice to prevent nuclease contamination. Wear gloves and use nuclease-free water and labware.
• Salts, buffers and organics often carryover from common nucleic acid purification techniques. The Slide-A-Lyzer MINI Dialysis Unit (Product #69550, 69560, 69570) may be used to dialyze off buffer, salt and organic contaminates from dilute purified nucleic acid in a quick 15-minute procedure. (Figure Mini microtube dialysis)
• See Nucleic Acid Quantitation button for determination of concentration and purity. (back to top)

Formula Formulas appear in the purification protocols under Notes/Conditions.(back to top)

1. Chomczynski, P. and Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction. Anal. Biochem. 162, 156-9.
2. Laird P.W., et al. (1991). Simplified mammalian DNA isolation procedure. NAR, 19: 15, 4293.
3. Maloy, S.R. (1990). Experimental Techniques in Bacterial Genetics. Jones and Bartlet: Boston.
4. Sambrook, J., et al. (1989). Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Press: New York.(back to top).

Amicon-Millipore
The Ultrafree-DA DNA device is based on the same principal as the figure X only a nebulizer chops up the gel slice before it is filtered. Purifies DNA 100bp to 100kb http://www.millipore.com

Bio-Rad
Filtration devices similar to the Amicon units.
http://www.biorad.com

Sigma-Aldrich
GenElute filtration devices that trap agarose and remove EtBr. A 10-minute procedure that requires no melting or washing steps. Recovery is reported to be 30-70% for 50bp-23Kb. Recovery decreases with increased DNA fragment size.

Qiagen
Offers QIAEX II Gel Extraction Kit and QIAquick gel extraction kit. The QIAEX II gel extraction kit works with a washable pelletable sillica resin. The QIAquick gel extraction is a spin column with a silica high salt binding resin. In both kits, the gel slice is dissolved prior to binding to the column and the DNA is bound with a high salt binding resin so that you can elute the nucleic acid in nuclease-free water or TE buffer instead of having it in TBE or TAE electrophoresis buffer. Both kits purify nucleic acids 40bp to 50kb. For QIAEX II Gel Extraction Kit:
http://www.qiagen.com/catalog/chapter_09/chap9b.asp
For QIAquick Gel Extraction Kit:
http://www.qiagen.com/entry.asp?URL=/catalog/chapter_09/chap9b5.asp

Roche-Boehringer Mannheim
The gel slice is dissolved and the nucleic acid is bound to a silica resin via high salt. The researcher may elute the nucleic acid in TE or water. Roche claims 80% or greater recovery.

Slide-A-Lyzer MINI Product Nos. 69550, 69560, 69570.
Slide-A-Lyzer MINI Float 69588