Lab 2: Genomic DNA prep
There are several different protocols for genomic DNA preparation. The one used in this lab lyses cells using a combination of Proteinase K (from the fungus T. album) and the detergent sodium dodecyl sulfate (SDS). (EDTA is also included to inactivate DNAases.) Proteins are removed from the solution using phenol/chloroform extraction, and DNA is concentrated by ethanol precipitation. This protocol works well for preparing genomic DNA from animal tissues, although solid tissues will have to be minced to small fragments before digestion (using a chilled mortar and pestle). RNAase treatment may also be used to remove RNA from DNA preps.
Phenol is used to denature protein in nucleic acid preps. The denatured protein precipitates out at the interface between the DNA-containing aqueous (top) layer and the phenol (bottom) layer. When removing the aqueous layer, it is important to make sure that none of the material at the interface is transferred. This results in an unavoidable loss of some of your nucleic acid sample. If you have a small volume of DNA sample, losses can be reduced by diluting your sample out to a larger volume – this will reduce the fraction of your sample that is lost with the interface. Alternatively, the phenol interface can be re-extracted with 100 microlitres of TE buffer following removal of the aqueous layer. The re-extracted DNA can be added to the first extraction before the chloroform extraction step. Because phenol is partly hydrophilic, water will migrate into the phenol layer, and reduce the volume of the sample. This can be minimized by saturating the phenol with Tris buffer before use.
Chloroform is used to remove phenol and remaining denatured proteins from nucleic acid preps. The chloroform is mixed with isoamyl alcohol to improve separation between aqueous and organic phases.
DNA is concentrated by precipitation with ethanol or isopropanol in the presence of 0.1 to 0.5 M monovalent cation solutions (e.g. ammonium acetate, sodium acetate, NaCl). The yield of DNA can be improved by using cold 95 % ethanol, but this may also precipitate excess NaCl from solution. The precipitation is usually followed by a “wash” step using 70 % ethanol, which removes excess salt precipitated with the DNA. Precipitation of DNA may be carried out in two ways. The first way involves layering ethanol on top of the DNA solution, and carefully winding the precipitated DNA onto a pasteur pipette with a tip melted into a U shape. This gives a better yield of undamaged high molecular weight DNA, which may be important for preparing genomic DNA libraries. For most purposes, (e.g. Southern blots and PCR), DNA is precipitated by mixing the aqueous phase with the ethanol by gentle inversion, followed by centrifugation. Some kits substitute DNA adhesion to powdered glass or diatomaceous earth for ethanol precipitation steps.
Once the DNA has been precipitated and washed, it must be dried to remove ethanol, which will interfere with restriction endonuclease digests. This can be done by air-drying or by drying under vacuum. If drying by vacuum, care must be taken not to completely desiccate the sample – this will make it difficult to resuspend high molecular weight DNA.
When preparing genomic DNA from plants or bacteria, polysaccharide contamination may be a problem. (Polysaccharides often interfere with restriction endonuclease activity.) Polysaccharides can be removed from DNA preps using the chemical cetyltrimethylammonium bromide (CTAB) – see Murray and Thompson, 1980.
Another type of protocol involves boiling cells in the presence of Chelex resin (Biorad). The resin chelates the Mg2+ cofactors required by DNAase enzymes, protecting DNA from degradation. See Walsh et al., 1991.
Precautions:
Phenol and chloroform will be used in this lab. Both chemicals must be handled in the fume hood, using gloves, to prevent skin contact or inhalation. Phenol and chloroform may penetrate gloves – so discard contaminated gloves immediately. Notify TA about spills. Read MSDS sheets for further information.
Materials and Methods
Materials
Oncorhynchus mykiss (rainbow trout) blood
Buffer A 0.1 M NaCl
10mM MgCl2
20 mM Tris-HCl pH 7.5
Proteinase K solution:
5 mg of Proteinase K dissolved in 7 ml of Proteinase K buffer
preincubated at 65ºC for 30 minutes
Proteinase K buffer: 0.2 M NaCl
50 mM EDTA
0.1 M Tris pH 8.0
1 % SDS
phenol (saturated with 10 mM Tris-HCl, pH 8)
chloroform/isoamyl alcohol (24:1)
95% Ethanol (– 20 ºC)
70% Ethanol (Room temperature
TE buffer (10 mM Tris pH 7.5, 1 mM EDTA)
Methods
Week 2:
1. Add 100 microlitres Buffer A to 5 microlitres fish blood
2. Add 600 microlitres Proteinase K solution. Incubate at 65ºC for 30 minutes.
3. Melt the tip of a pasteur pipette into a “U” shape. Leave to cool.
4. Add 0.5 ml phenol, firmly cap tube, and mix by inverting the tube several times. Centrifuge 5 minutes on microfuge. (Sample processing will be more efficient if 4 groups share a microfuge at once.)
5. Transfer upper (aqueous) layer to fresh tube. (Use a p1000 and a blue tip with the bottom cut off.) Leave all of the white material at the interface (denatured proteins) behind – you will lose some of the aqueous layer in this process.
6. Add 0.5 ml chloroform/isoamyl alcohol (24:1), mix and centrifuge as before. Transfer upper (aqueous) layer to fresh tube, leaving interface behind.
7. Estimate the volume of the remaining aqueous solution. (This can be done using a p1000). Add 2 volumes of cold 95% ethanol. Do not mix immediately. Use the pasteur pipette with the U-shaped tip to try to collect DNA from the interface between the ethanol and aqueous solutions. Return the DNA to solution, and mix the solution gently by inversion. Precipitate the DNA for 15-30 seconds in a microcentrifuge. Make sure that the hinge of the Eppendorf lid is in a consistent place.
8. Remove supernatant with a p1000. Wash pellet with 70% ethanol. (This can be done with gentle inversion – try not to resuspend the pellet.) Precipitate the DNA for 15-30 seconds in a microcentrifuge.
9. Remove supernatant with a p1000. Pulse spin on microcentrifuge to collect remaining liquid at bottom of tube. Remove with p200. Allow DNA to air-dry 5-10 minutes. (When you cannot smell ethanol any longer, the pellet is dry enough.)
10. Resuspend pellet in 100 microlitres of TE buffer. Place DNA sample on rack provided. Sample will be stored at 4ºC until next week.
Week 3:
Estimate the concentration and purity of DNA prep by measuring absorbance at 260 and 280 nm. Measurements must be done using quartz cuvettes – these are fragile and expensive, so handle with care and avoid scratching them.
Absorbance measurements should be performed using a DNA solution with an absorbance in the range between 0.05 and 0.5 absorbance units. A 1/20 dilution – (50 microlitres DNA solution in 0.95 ml TE) will usually be in this range. If it is not, try different dilutions. A pure DNA solution will usually have an A260 nm to A280 nm ratio of >1.8 (although various other molecules in solution may lower this). An A260 nm to A280 nm ratio of 1.5 usually indicates equal amounts of protein and DNA in solution.
If the A260 nm to A280 nm ratio is >1.8, DNA concentrations can be estimated using the standard 50 micrograms/ml DNA = 1.0 A260 nm unit.
References:
Murray, M.G. and Thompson, W.F. (1980). Nucleic Acids Research 8: 4321.
Walsh et al. (1991). Biotechniques 10: 506-513.
Study questions (due in 4th week of labs) – show samples of all calculations
1. Calculate the A260 nm to A280 nm ratio for your sample.
2. Calculate the nucleic acid concentration in your sample.
3. Calculate the yield of nucleic acid (micrograms of nucleic acid/ml blood sample).
4. Could the protocol used in this experiment be used to isolate DNA from other tissues? What modifications would be needed to prepare DNA from testicles? What modifications might be necessary for preparing DNA from human tissue?
5. Why was DNA resuspended in TE buffer?
6. Why was the end cut off of the p1000 tip used for transfer of genomic DNA solutions?