Main Title Image

Last updated

August 10, 2003
 
IntroductionResearch PagesPeople involvedPhoto galleriesPublicationsOther sample sitesEducationLinks to other micobial observatories
DNA Extraction Protocol #2 [back]  
 

This procedure was originally developed for Listeria monocytogenes but has worked well with other Gram+ bacteria


1. Pellet cells from 10 ml overnight cultures in BHI or LB and wash in 5 ml of 0.1X SSC.
2. Resuspend in 1 ml 0.01 NA-PHOSPHATE BUFFER (We have this buffer for protein purification, come to get) in 20 % SUCROSE (pH 7.0), add lysozyme to 2.5 mg/ml, and incubate at 37 C for 45 min or longer.
3. Add 9 ml LYSIS BUFFER (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 500 mg pronase B, 1 % SDS), and incubate additional 30 min (longer) at 37°C.
4. Phenol and chloroform extract lysed cells, and ethanol precipitate the DNA with 0.1 vol. 3 M sodium acetate, pH 4.8 and 2 vol. 95% ethanol.
5. Spool out DNA with a glass rod, wash once with 80% ethanol before drying.
(Or by centrifugation)


Some bacterial species may require a longer incubation in lysozyme. For Renibacterium salmoninarum (a G+ salmon pathogen we work with), lysozyme incubations overnight at 37 C worked very well with high yields of DNA

A. PHENOL/CHLOROFORM EXTRACTION OF DNA

Materials:
· phenol:chloroform (1:1)
· chloroform


1. Add an equal volume of buffer-saturated phenol:chloroform (1:1) to the DNA
solution.
2. Mix well. Most DNA solutions can be vortexed for 10 sec except for high
molecular weight DNA which should be gently rocked.
3. Spin in a microfuge for 3 min.
4. Carefully remove the aqueous layer to a new tube, being careful to avoid the interface. (Steps 1-4 can be repeated until an interface is no longer visible)
5. To remove traces of phenol, add an equal volume of chloroform to the aqueous layer.
6. Spin in a microfuge for 3 min.
7. Remove aqueous layer to new tube.
8. Ethanol precipitate the DNA


Phenol extraction of DNA samples


Phenol extraction is a common technique used to purify a DNA sample. Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded, including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.


Protocol


1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.
2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.
3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during the first phenol extraction.
4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3 minutes at room temperature. Remove and discard the upper, ether layer, taking care to remove phenol droplets at the ether:aqueous interface. Repeat the ether extraction.
5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate, as discussed below.


B. Concentration of DNA by ethanol precipitation


Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20°C overnight. To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.


Protocol


1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at -20°C overnight at this stage.
2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 minutes at 4°C, decant the supernatant, and drain inverted on a paper towel.
3. Add 80% ethanol (corresponding to about two volume of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above.
4. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about 5-10 minutes, or until dry.
5. Always dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA (termed 10:0.1 TE buffer).
6. It is advisable to aliquot the DNA purified in large scale isolations (i.e. 100 mg or more) into several small (0.5 ml) microcentrifuge tubes for frozen storage because repeated freezing and thawing is not advisable.
Notes on precipitation of nucleic acids


A. General rules


Most nucleic acids may be precipitated by addition of monovalent cations and two to three volumes of cold 95% ethanol, followed by incubation at 0 to -70 °C. The DNA or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at 4°C. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes residual salt and moisture.


The general procedure for precipitating DNA and RNA is:


1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be precipitated,
2. Add two volumes of cold 95% ethanol,
3. Place at -70°C for at least 30 minutes, or at -20°C overnight.


or alternatively


1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml of sterile water. Mix by inversion and store at -20°C.
2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be precipitated.
3. Place at at -70°C for at least 30 minutes or -20°C for two hours to overnight.
* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA also may be precipitated by addition of 0.6 volumes of isopropanol.


B. Notes on phenol extraction of nucleic acids


The standard and preferred way to remove proteins from nucleic acid solutions is by extraction with neutralized phenol or phenol/chloroform. Generally, samples are extracted by addition of one-half volume of neutralized (with TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for a few seconds to form an emulsion. Following centrifugation for a few minutes, the aqueous (top) phase containing the nucleic acid is recovered and transferred to a clean tube. Residual phenol then is removed by extraction with an equal volume of water-saturated diethyl ether. Following centrifugation to separate the phases, the ether (upper) phase is discarded and the nucleic acid is ethanol precipitated as described above.


A 1:1 mixture of phenol and chloroform also is useful for the removal of protein from nucleic acid samples. Following extraction with phenol/chloroform, the sample should be extracted once with an equal volume of chloroform, and ethanol precipitated as described above.


 
   

This website is maintained by Alam's Lab, Department of Microbiology, University of Hawai'i. All rights reserved.

Link to National Science Foundation website Link to University of Hawaii website Link to Department of Microbiology website Link to Maqsudul Alam lab website Back to main page