Alternative Procedures
Unless a specific announcement has been made in the D2L News Area, assume that you will not be doing any of the procedures below.
Phenol-chloroform extraction (if spin columns are unavailable)
7. Alternative procedure if spin columns are unavailble. Transfer the supernatant to a fresh tube, add an equal volume (440 μl) of phenol:chloroform:isoamyl alcohol (25:24:1) and vortex vigorously for 5-10 seconds. Read the Safety and Disposal instructions at the bottom of this page before handling chloroform. Centrifuge for 3-5 minutes in a microcentrifuge at maximum speed to achieve phase separation. Carefully transfer about 430 μl of the top aqueous phase to a clean tube carefully avoiding any precipitated proteins at the aqueous-organic interface. Optional: If chloroform is accidentally transferred over into the clean tube, add 100 μL of chloroform (without phenol), centrifuge for 1 minute and carefully remove the aqueous phase a second time into a clean tube.
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What's going on? Phenol:chloroform is very effective at extracting proteins, such as DNase, RNase, lysozyme and all of the proteins from the lysed cell. These contaminants will become insoluble in the aqueous phase, and will be found at the aqueous-organic interface. This step also removes the RNA monomer products of RNase. Isoamyl alcohol is added to prevent foaming at the interface. |
8. Centrifuge for 3-5 minutes in a microcentrifuge at maximum speed to achieve phase separation. Carefully transfer about 430 μl of the top aqueous phase to a clean tube carefully avoiding any precipitated proteins at the aqueous-organic interface. Optional: If chloroform is accidentally transferred over into the clean tube, add 100 μL of chloroform (without phenol), centrifuge for 1 minute and carefully remove the aqueous phase a second time into a clean tube.
10. Mix the aqueous solution with 1000 μl of pure ethanol. Let sit 2 minutes at room temperature to precipitate nucleic acids.
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What's going on? Ethanol is less polar than water. Ethanol disrupts the screening of charges by water. At a 65-70% ethanol, the electrical attraction between phosphate groups and the cations from the prior steps becomes strong enough to form stable ionic bonds and DNA precipitates as a neutral solid. |
11. Centrifuge for 5 minutes in a microcentrifuge at room temperature at maximum speed to pellet the plasmid DNA.
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What's going on? Many procedures recommend DNA precipitation and centrifugation at cold temperature, which is typically associated with decreases the solubility of chemicals. However, decreasing the temperature of an alcohol-aqueous solution increases its dielectric constant.1 As a result, DNA precipitation efficiency is in fact diminished (particularly below 0 °C) and other salts co-precipitate. The cleaner the DNA, the more difficult it will be to see the pellet. This protocol produces plasmid DNA with sufficient purity for many procedures, and impurities are high enough so that the pellet should be readily visible. |
12. Discard the supernatant and wash the pellet by adding 1 mL of 70% ethanol and do not mix. Centrifuge for 1 minute if the pellet was dislodged. Discard the supernatant into a clean tube and check that the pellet was not lost. Repeat.
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What's going on? This step washes away salts and other small molecules, particularly phenol. In practice, we have found that significant phenol is present after one wash. |
13. Remove the supernatant as completely as possible and let the pellet dry in the SpeedVac for 10-20 minutes.
14. Dissolve the pellet in 20 μL of TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).
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Measurement of DNA concentration with standard UV-Vis spectrophotometer
1. Dilute the plasmid DNA, 2 μL into 500 μL of TE buffer.
2. Transfer some of the solution to a UV transparent cuvette. Quartz is UV-transparent, but many plastics are not, ask your instructor to make sure you are using the correct cuvettes. Determine the A260, A280, and A230 using a cuvette containing TE as reference. The reading should be in the instrument's linear range 0.01 - 2.0 (for the Beckman Coulter DU 700). If not, you must quantitatively dilute the sample until it is within range and measure A260 and A280 on the new dilution. If the A260/A280 ratio is higher than 1.8, check the A260/A230 ratio.
3. Calculate your DNA concentration as [DNA] (ng/μL) = A260 × 50 (see introduction). Keep in mind that contaminating chromosomal DNA, RNA, phenol, and other organics may contribute to the results from this measurement. You will be able to see contaminating nucleic acids on the agarose gel.
4. To estimate your actual DNA concentration, multiply the value from the last step by your dilution factor and multiply by the % purity of your DNA as estimated from your absorbance ratios using the plot in the introduction.
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