General Techniques for Handling Nucleic Acids
In general nucleic acids are relatively easy to work with. Unlike proteins which lose their enzymatic activities very easily, as long as you pay attention to a few important details, nucleic acids are stable. DNA is very stable, while RNA needs a bit more attention to detail. The primary concern is nucleases. For this reason all buffers, pipets, pipetman tips, tubes and anything else that will come into contact with the DNA or RNA should be autoclaved and subsequently handled as a sterile solution. Because fingers are an excellent source of nucleases, you should be careful not to touch anything with bare fingers. Again, you need to be more careful when working with RNA than with DNA. Samples should be kept on ice whenever possible. RNA is best stored frozen, preferably at -70o C. DNA is usually stored frozen at -20o C or at 4o C. DNA should always be stored in TE (10 mM tris-HCl, pH 8.1, 1 mM EDTA). Since most nucleases require magnesium for activity, the EDTA helps inactivate them. RNA is usually stored in distilled water which has been treated with diethyl-pryocarbonate (DEP) to inactivate RNAses. Many RNAses survive autoclaving, so DEP should be used to treat solutions whenever possible.
DEP treatment of solutions.
Make solutions 0.1% DEP, shake, then autoclave the solution. While the solution is still hot -- but not while it is superheated -- shake the solution again. DEP is inactivated by water and heating. It is important to be sure that DEP has been inactivated as it will modify RNA and inactivate enzymes such as reverse transcriptase (used to make cDNA) or kinase (used to label RNA in vitro). For RNAs which will be used for cDNA synthesis it might be desirable to avoid DEP completely. DEP is also highly toxic to humans and should always be handled in a chemical hood. Tris, perhaps the most common buffer for nucleic acid biochemistry is incompatible with DEP, so the best thing for tris solutions is to use DEP treated water when making tris solutions, then autoclave the tris again after addition.
Phenol is used to denature and remove proteins from nucleic acid solutions. Phenol is also quite efficient at removing proteins from your skin, so be sure to wear gloves whenever using phenol. Do not use any tube which is clear. Phenol (and chloroform) will almost certainly melt it!
1. Add an equal volume of phenol to the sample to be deproteinated then shake or vortex to mix.
2. Spin 2 minutes in a microfuge or 10 min at 8,000 RPM in a Sorvall RC-5 or Beckman J2-21. There will be two phases, the phenol (usually on the bottom) and the aqueous (usually on top). Since we have put an anti-oxidant into the phenol, it will be yellow in color. Between the phenol and aqueous phases you may see a white interface layer. This is the denatured proteins.
3. Carefully remove the aqueous phase (which contains the DNA) and transfer it to a fresh tube. Try to avoid transfering any of the denatured protein (the white material at the interface of the phases). Repeat the extraction of the aqueous phase until there is no longer any material at the interface.
4. Generally phenol extractions are followed by 2 extractions with chloroform containing 4% isoamyl alcohol. Again, equal add an equal volume of chloroform and mix thoroughly. This step helps remove residual protein, as well as phenol which might remain from the phenol extraction.
5. Centrifuge and recover the aqueous phase (usually the top phase). This helps remove any remaining proteins and also helps remove the phenol.
6. Finally, if the DNA is to be manipulated enzymatically in a subsequent step, it may be helpful to extract a couple of times with ethyl ether. Since ether has such a low density, the aqueous phase (which contains the DNA) will be on the bottom. Ether extraction will remove residual chloroform and phenol. Residual ether is easily removed by heating briefly at 37o C.
Following these treatments it is usually a good idea to ethanol precipitate the DNA or RNA.
To prepare phenol, crystalline phenol is warmed to melt it and extracted by shaking with 1 M tris pH 8.1, 20 mM EDTA. The aqueous layer is removed and replaced with fresh 1 M tris, 20 mM EDTA and the extraction repeated. After two or three such extractions, change the aqueous buffer to 20 mM tris pH 8.1, 1 mM EDTA and extract two or three more times. Store the phenol under this buffer. Add 8 hydroxy-quinoline (an antioxidant) to 1%. This also turns the phenol yellow which facilitates following the phenol phase during extractions.
Ethanol Precipitation of Nucleic Acids
Ethanol precipitation is an easy way to concentrate nucleic acids, or to transfer them into a new set of buffer and salts.
1. Add 0.1 volumes 3 M NaOAc, pH 5.5, or 1 volume of 4 M NH4OAc. Mix well. Usually the NaOAc is used. It is said that small molecular weight contaminants do not precipitate as well when NH4OAc is used, consequently it is often used when the goal of precipitation is to remove nucleotides, etc.
2. Add 2 volumes of absolute ethanol which has been chilled to -20o C (most easily by storing in the freezer), or 4 volumes of ethanol if you used NH4OAc as the salt.
3. Incubate the ethanol precipitation at -20o C for 6 hours, or at -70o C for 30 min, or in a dry-ice ethanol bath or liquid nitrogen for 5 to 10 minutes.
4. Allow the tube to warm to room temperature and centrifuge 10 min in a microfuge, or 10 min at 10,000 RPM in a Sorval or Beckman high speed centrifuge. Small amounts of DNA are recovered more efficiently if you spin the precipitation 20 to 30 minutes. If you have very small amounts of DNA or RNA in a large volume you may want to centrifuge 30 minutes at 25,000 RPM in an ultracentrifuge.
5. Carefully remove the liquid from the tube following centrifugation and add room temperature 70% ethanol to the tube, and centrifuge again. This washing step helps remove any residual salt and is very useful.
6. Dry the pellet briefly in a vacuum. Be careful to not go too long. Sometimes too much drying will make the pellet difficult to solubilize. The pellet now contains relatively salt-free DNA or RNA which can be solubilized in the buffer of your choice.
PEG Precipitation of DNA
This procedure is good for cleaning up DNA samples before doing sequencing reactions.
1. Add 1/4 volume of 4M NaCl and mix well.
2. Add equal volume ( starting volume) of 13% PEG.
3. Incubate on ice for 1/2 hour or longer.
4. Spin 15 min and remove supernatant.
5. Add 0.5 ml 70% EtOH, spin again and remove supernant.
6. Dry pellet in Spin-Vac.
Quantitation of DNA and RNA
DNA and RNA are very easily quantitated by simply measuring the absorbance of the solution at 260 and 280 nm wavelength. Because this is in the ultraviolet range, it is important to be sure that the cuvettes you use are UV transparent (usually that means they're made of quartz instead of glass or plastic). Pure nucleic acids should show a A260/A280 ratio of greater than 1.75 - 1.80. Values lower than this imply contamination with lipid or protein.
An A260 of 1.0 corresponds to a concentration of 40 m g/ml for RNA or 50 m g/ml for DNA.
Restriction Enzyme Digestion
Reference: Maniatis et al., Molecular Cloning p. 104
To avoid contaminating buffers and expensive enzyme stocks, it is very important that all tubes, pipette tips and solutions be autoclaved before use. Because of nucleases on your fingertips, be careful not to touch pipette tips with your fingers.
Each restriction enzyme has optimal reaction conditions, as specified by the manufacturer. To avoid making up many different buffers, the 3 buffer systems below can be used for most enzyme digestions. These buffers can be made as a 10X stock and stored in aliquots at -20o C.
These represent 1X concentrations.
(1) Some enzymes have unusual requirements, such as for ammonium sulfate or unusually high salt.
(2) To stabilize the enzyme during the reaction, you may wish to add 100
While most enzymes work in the above buffers, it is critical to recognize that an enzyme produced from one company may not cut in the buffer of the same enzyme produced from a different company. The lab purchases enzymes from at least 4 different sources, so always be aware of which company's enzymes you are using.
A restriction enzyme unit is defined as the amount of enzyme needed to digest 1mg of DNA to completion in 1 hr under the specified conditions. In cases where rapid digestion is desired or where complex digestion patterns are expected, you can use a 4-5 fold excess of enzyme. Since glycerol concentrations over 5% in the final reaction can sometimes inhibit the enzyme, try to avoid using enzyme volumes larger than 1/10 of the reaction volume.
Setting up the reaction:
In a 0.5 or 1.5 ml Eppendorf tube, set up reactions in a total volume of 10-20ml. Mix the DNA, 10X buffer, and water to bring the reaction to the final volume. Carry the enzyme from the freezer to your bench on ice. Using a capillary pipette, or Pipetteman with a fresh, autoclaved tip, carefully pipette the enzyme into your reaction. Return the enzyme immediately to the freezer. Gently mix the reaction and incubate at the proper temperature (usually 37o C.) for 1-2 hr. To stop the reaction, heat the sample at 65o C for 10 min. Spin briefly in the microfuge to make sure the entire reaction is at the bottom of the tube. If the sample is to be analysed on a gel, add 1/10 volume of 10X Loading dye. If the sample is to be used for ligation or other processing, be sure not to add dye. It only goes into samples to be run on gels. Digested samples may be stored at -20o C before running the gel.
10X Loading Dye:
100 mM EDTA
50% glycerol (or 25% ficoll 400)
1% SDS (optional)
0.25% bromphenol blue
Submarine Agarose Gels
Southern Transfer of DNA Gels
Random Primed Synthesis for the Preparation of Labeled DNA Probes
1. Take up DNA to be labelled in 21ml dH2O.
2. Heat denature the DNA at 90oC for 15 min, then 37oC for 5 min.
3. Set up the following reaction:
a. 21ml heat denatured DNA (25-50 ng DNA).
b. 3ml NTR -A buffer.
c. 5ml [32P]-dATP.
d. 1ml 30 OD units/ml stock of pd(N)6 random primers
e. 1ml Klenow.
4. Incubate at RT for 3 hrs or at 37oC for 30 min.
5. Stop reaction by adding 70 ml of TE with an additional 2 mM EDTA plus 0.25% SDS added.
6. Make duplicate DE81 filters with 1 ml of the reaction on each. After drying, wash one as usual in 0.4 M Na2HPO4. Count in aquasol.
7. Prepare spin column to remove unincorporated counts as follows:
a. place glass wool in bottom of 1 ml syringe and fill with G50 sephadex in buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.3 M NaCl, 0.05% SDS).
b. spin column for 4 min. at maximum speed in tabletop centrifuge.
c. wash 2 times (2 minutes at maximum speed) with 100 ul buffer, making sure you recover 100 ml after last spin.
d. load your labelling reaction on the column, spin 2 min. at maximum speed.
e. count 1 ml of recovered sample.
An alternative to running a spin column is to ethanol ppt. the reaction with 0.1 volume salt, 2 volumes ethanol to remove unincorporated counts.
By keeping the reaction volume to 20 ml instead of 30 ml, the concentration of dATP is increased by 50%. This may allow greater incorporation of label.
Alternatively, Sephadex G-50 can be packed into a 1.0 ml tuberculin syringe.
1. Plug the syringe with a bit of siliconized glass wool. Fill the barrel of the syringe with G50 slurry and allow it to settle. Continue to add slurry until the barrel is full of settled G50.
2. Place the syringe in a 15 ml conical centrifuge tube and spin for 2 minutes at 2000 RPM. Repeat the process twice, adding buffer to the column each time, to equilibrate the column. Remove any fluid from the 15 ml conical tube or replace it with a new one.
3. Now add the reaction mixture (volume should be less than 100
Filter Hybridization Methods
Hybridization can be performed in our hybridization oven, or, if it is unavailable, you can use a seal-a-meal bag. The hybridization oven offers several advantages. First, you have greater control over the temperatures. Second, because the hybridization solution is constantly mixed by the rolling of the hybridization tubes in the hybridization oven problems with uneven hybridization are reduced.
Place the filter along the inside of the roller bottle being sure to avoid having large bubbles trapped between the filter and the glass walls of the roller bottle. The minimum volume of hybridization solution for the roller bottles is about 10 mls.
Place nitrocellulose filters in a seal-a-meal bag. Because the filters are very fragile, one approach is to cut the bag open on 3 sides and lay it out flat. Then position the filters on one side and then fold the other side over the filter. Seal the bag on 3 sides, close to the filter so that the buffer will be concentrated around the filter. If the filter is dry, it is usually possible to simply slide it into a bag, and then to seal around it. Pure nitrocellulose (vs nylon based membranes) tends to swell once in solution so don't cram it in.
DNA-DNA Hybridization Solution:
5X Denhardts solution
10 ml of 50X stock*
30 ml of 20X stock
2.5 ml of 20% stock
20 mM NaPO4, pH 6.5
4 ml of 0.5M stock
10mg/ml salmon sperm DNA
0.1 ml of 10 mg/ml
dH2O to 100 ml
*This solution can be stored frozen and thawed by incubation at 65o C.
DNA-RNA Hybridization Solution
50% deionized formamide
50 ml formamide
5 X SSC
25 ml of 20X stock
50 mM NaPO4, pH 6.5
5 ml of 1M stock
1 ml of 10% stock
5X Denhardt's solution
10 ml of 50X stock
10-100mg/ml salmon sperm DNA
1.0 ml of 10 mg/ml
10-50 ng/ml poly A (optional)
H2O to 100ml
This solution can be stored at -20oC and thawed at time of use.
Riboprobe Hybridization Solution
50ml of stock
30ml of 20X
10ml of 50X
1 mM EDTA
0.1ml of 1M
25 mM NaPO4
2.5ml of 1M
200mg/ml Salmon sperm DNA
H2O to 100ml
This solution can be stored at -20oC
1 g polyvinylpyrolidone
1 g BSA
1 g Ficoll
dH2O to 100 ml
Salmon Sperm DNA
1 g Salmon Sperm DNA
100 ml H2O
Mix sperm DNA in water and autoclave. Store at 4oC
Add 10 to 20 ml of hybridization solution to the roller bottle. Be sure that there are no bubbles trapped between the filter and the wall of the roller bottle. Place into hybridization oven set for the desired temperature. Use 65o C for DNA-DNA and 37 - 42o C for RNA-DNA hybridizations. Pre-hybridize for at least 3 hr, overnight is fine. For using ribo probes the procedure is a little different. Prehybridize for 10-20 minutes at 50oC.If you are using a seal-a-meal bag, add 10-20 mls hybridization solution and seal, trying to avoid leaving bubbles in the bag. Leave enough room on this seal so that the bag can be cut open and sealed again later. Double-check the seals around the bag. Place the bag into a waterbath.
Carry out the following steps involving radioactivity on absorbent paper. In an eppendorf tube, mix the labeled probe (about 1-2 million cpm/ml of hybridization solution) with 1/10 volume of 1 N NaOH. Boil the tube 10 min then chill on ice. Add 1/10 volume of 1 N HCl to neutralize. Add the probe to the appropriate volume of fresh hybridization solution. Carefully pour or pipette out the prehybridization solution and replace with the hybridization solution. Boiling the probe is essential. If you don't denature the probe DNA, it won't hybridize to anything! If you are using a hybridization bag, cut open one edge of the bag and squeeze out all the prehybridization solution. For a filter 10 cm by 10 cm, 5 ml should be enough. Seal the bag, again avoiding bubbles. Wipe the bag carefully to remove any label which may still be on the outside of the bag, then return to the waterbath. When using riboprobes the procedure is identical except that the probe is not boiled and 50oC is used.
Hybridize 12-48 hr.
WASHING THE FILTERS
DNA/DNA and DNA/RNA
Prepare 1 liter of wash buffer -- 2 X SSC, 0.1% SDS for DNA-DNA, and 0.1X SSC, 0.1% SDS for RNA-DNA. Remove the hybridization solution and place in the appropriate liquid radioactive waste container. Rinse the hybridization bottle two or three times with wash buffer and discard the waste to the radioactive waste container. Add 10-25 ml wash buffer and return to the hybridization oven. If using bags, cut open the hybridization bag and carefully squeeze the radioactive hybridization solution onto absorbant paper such as a blue diaper or benchcoat. Alternatively use a pasteur pipette to remove the labeled hybridization solution to the appropriate container for liquid radioactive waste. Carefully cut open the rest of the bag, taking extreme care to avoid spreading label everywhere -- work over absorbant paper and wear gloves, a lab coat and your radiation badge. Place the filter directly into a plastic box containing enough room temperature 2X SSC to cover the filter with a good layer of solution. Swish the filter around and discard the now radioactive solution into the radioactive waste. Repeat the 2X SSC rinse at room temperature. Take great care to avoid having the filter dry out even momentarily. This will help minimize problems with background.
From now on, the wash solutions can be poured down the sink. Wash the filter 4 times at the desired temperature (60oC for DNA-DNA hybridizations and 50o C for RNA-DNA hybridizations) for 1/2 hr in 250 mls. Place the filter on 3MM paper to dry and then wrap in saran wrap. The filter is now ready for autoradiography.
Remove filter as above. Wash 2X 30 minutes at room temperature with 1X SSC/0.1% SDS. Wash 2X 30 minutes at 65C with 0.1X SSC/0.1% SDS. It may be necessary to RNase treat the filters to remove background. Put filter into 2X SSC with 1mg/ml RNase A at RT for 1 minute. Wash 1X 30 minutes with 0.1SSC/0.1%SDS.
Guanidine Thiocyanate procedure for RNA extraction.
Reference: Chirgwin, et al. 1979. Biochemistry 18, 5294.
This procedure is a very general one. It will work for virtually any cell or tissue type. Below is a description of the particular version for Dictyostelium. On the next page is a description for frozen tissue.
1. Harvest cells, wash once in PDF.
3. Resuspend pellet (containing 1 to 5 X 10
4. The RNA is next separated from cellular proteins and DNA by centrifugation in a CsCl gradient. Rinse SW28 polyallomer tubes sequentially with GuSCN and DEP H2O. Pre-rinse a pipet with GuSCN. Pipet 15 ml of 5.7 M CsCl into each tube. Very carefully layer the cell lysate on top of the CsCl, taking care to avoid disturbing the interface. Centrifuge at 22,000 RPM for 22 hours, at 22OC. Smaller samples (up to 108 cells) can be lysed in 6 mls GuSCN, layered on a 5 ml cushion of CsCl and spun for 7 hours in the SW41 rotor.
5. After centrifugation, the proteins will be near the interface between the CsCl and the guanidine thiocyanate. The DNA should be visible as a band about 1/3 of the way up the tube from the bottom, and the RNA is a pellet at the bottom of the tube. Carefully aspirate the solution down to below the DNA band. Then pour off the remaining liquid and keep the tube inverted. This prevents any potential contamination of the RNA with material from higher in the gradient.
6. Cut the top of the tube off using a razor blade, and great caution. Several people have cut themselves during this part of the procedure.
7. Add 0.5ml of 70% ethanol. Using an autoclaved pipet tip dislodge the pellet and transfer it and the 70% ethanol to an autoclaved 1.5 ml microfuge tube.
8. Rinse the centrifuge tube with 0.5ml 70% ethanol and transfer this rinse to the microfuge tube. Be sure to transfer any little bits of the pellet which may remain.
9. Pellet RNA by spinning 10 minutes in microfuge. Large pellets should be split into several tubes.
10. Remove ethanol and add 1.0 ml 70% ethanol for a second wash.
11. Pellet RNA again and carefully remove all drops of ethanol. You may need to dry it in the speed vac.
12. Resuspend pellet in 50 ml DEP H2O (or 100 ml if there's a fairly large amount of RNA). Heat at 70OC for 30' to dissolve the pellet.
13. Determine the concentration by adding 2ml of the RNA to 0.5 ml of dH2O and reading the A260 and A280. RNA concentration is the A260 times 40 mg/ml. The 260/280 ratio should be better than 1.8, and is usually above 2.0. You now have relatively pure RNA.
Preparation of frozen tissue for RNA Extraction
1. Tissue should be cut into small pieces no larger than 1 cm3 and placed into liquid nitrogen in a 50 ml Corning tube immediately upon dissection. Store the tissue frozen at -70o.
2. Grind up tissue with polytron as follows. Set up polytron and rinse it with water (regular distilled water is fine for this). Get tubes of tissue one at a time from the freezer. Immediately add 15 ml of GuSCN to the tube and polytron until totally ground. Rinse polytron twice with dH2O between tubes. Do NOT place tubes on ice.
3. Spin tubes for 4 min. at full speed to spin down foam. Add additional GuSCN to 20 ml final volume.
4. Proceed with step 4. on previous page.
GuSCN LYSIS BUFFER
To make 100 mls: (Six tubes requires at least 150ml)
50 g guanidine thiocyanate (or isothiocyanate, they're actually the same thing).
0.5 g sarcosyl
5.0 ml 0.5 M EDTA, pH 7.50
Warm to solubilize, then filter to remove any particulate matter. Depending on the particular lot of guanidine, this filtration step may not be necessary.
5.7 M CsCl Solution
To make 100 mls:
dissolve 95.96 g CsCl in 50 mM NaOAc, pH 5.5/10 mM EDTA
Filter this solution with a #3 Whatman filter, then add 100 ul DEP, shake and autoclave. To avoid RNA pellets containing crystalline CsCl, it is advisable to check the refractive index of the 5.7 M CsCl solution after autoclaving. The refractive index should be 1.3998. If necessary, adjust with DEP treated dH
Isolation of RNA by Phenol Extraction.
Often a more rapid procedure for the isolation of RNA from many samples, or from small amounts of tissues. The following represents an alternative.
1. Pellet cells (<5 X 108)in an eppendorf tube.
2. Add lysis buffer (see below) and vortex until pellet is completely dissolved. If using tissue, be sure that the tissue is quickly dissolved, using a homegnizer or polytron.
3. Immediately add an equal volume of phenol and vortex.
4. Add 1X volume Chloroform/isoamyl alcohol (24:1) and vortex.
5. Heat to 550 for 5 minutes in a water bath. For rapid removal of protein it is critical that the tubes are as fully immersed in the water as possible.
6. Cool on ice for 5 minutes.
7. Spin in a microcentrifuge for 2 minutes.
8. Remove aqueous phase and repeat extraction with phenol/chloroform/IAA (without heating step).
9. Continue extractions with 24:1 chloroform/isoamyl alcohol until there is no protein precipitate at the interface (this usually requires only 2 extractions when the heating/cooling step is used).
10. Add 0.1 volume 3 M NaOAc and ethanol precipitate with 2.5 volumes ethanol. It is convenient at this point to have the aqueous RNA solution measure 0.4 mls so that 1 ml of ethanol can be added to the eppendorf tube.
11. Resuspend the pellet, which at this point contains both RNA and DNA, in 0.5 mls DEP treated dH2O. Incubate at 700 until dissolved. If the RNA is to be used for blotting only then is it can be dissolved in DEP treated 0.5% SDS to provide further protection from nucleases. At this stage the RNA is pure enough for northern blotting and can be quantified by absorbance at 260 nm. If further purification is desired this can be done as follows:
12. After cooling the solution on ice, precipitate the RNA with an equal volume DEP treated 8 M LiCl. This solution can be left overnight at 40 or immediately frozen in liquid nitrogen.
13. After thawing, pellet the RNA in the microfuge (15 minutes).
14. Remove as much LiCl as possible and resuspend the RNA in 0.4 mls DEP treated dH2O. The LiCl step leaves most of the DNA in the supernatant.
25 mM EDTA
10 mM TRIS pH 8.0
check the pH.
Add Diethylpyrocarbonate to 0.1% immediately before use.
This solution should be made from autoclaved stock solutions.
8 M LiCl:
33.9 g/100 mls H2O.
Add 0.1 ml DEP, shake and autoclave.
Isolation of Messenger RNA
Oligo-dT Cellulose Chromatography
Messenger RNA is readily purified by chromatography on oligo-dT cellulose. This procedure takes advantage of the fact that most cellular mRNA molecules have 3' poly(A) "tails". Total cellular RNA is run through a column consisting of oligo-(dT) bound to a solid support such as cellulose. The poly(A) tails basepair with the oligo-(dT) and are retained in the column while the remaining cellular RNAs (ribosomal, tRNAs, etc.) run through. The loading or "binding" buffer contains a high enough salt concentration that the basepairing between the oligo-dT and poly(A) is stabilized. When all the unbound RNA is washed through the column, the poly(A)+ RNA is eluted by reducing the salt concentration to a point which breaks the baseparing.
The following assumes a 1.0 ml bed volume of oligo-dT. Adjust volumes accordingly.
1. Prepare the column material by suspending the oligo-dT cellulose in binding buffer. One gram of oligo-dT generally has a binding capacity of 1.2 mg of poly(A)
2. Pour the slurry into a sterile plastic 5 or 10 ml disposable pipette which has been plugged with a bit of siliconized glass wool. Choose a pipette which leaves plenty of space above the bed of oligo-dT cellulose for the addition of buffers.
3. Treat the column with 5 column volumes of 0.1 M NaOH/5 mM EDTA. Occasionally the oligo-dT will turn yellow with the NaOH treatment. This color will wash away and should not be a concern.
4. Wash the column with 10 volumes of elution buffer. Check the pH to be sure the NaOH has all been washed out.
5. Wash the column with 10 volumes of binding buffer.
6. Resuspend the RNA to be used in 5 ml of DEP treated water. Heat to 65oC for 10 minutes.
7. Add 5 mls of 2X binding buffer and pour into column. It is generally a good idea to collect and save flowthrough until you know that you got poly(A)+ RNA.
8. Wash the column with 10 volumes of binding buffer.
9. Elute the poly(A)+ RNA with 1.0 ml elution buffer. Collect 0.5 ml aliquots into 1.5 ml Eppendorf tubes. Repeat 2 more times. Add 50 ml 3 M NaOAc, and 1.0 ml ethanol to precipitate RNA.
10. Collect precipitates by centrifugation in the microfuge and resuspend in DEP water at 1.0 mg/ml concentration or greater. Assume 2 to 5 % recovery of RNA put onto column.
11. A single pass over oligo-dT cellulose will not remove all of the ribosomal RNA. If greater purity is required, the RNA can be run through the column a second or even third time.
20 mM Tris-HCl, pH 7.6
0.5 M NaCl
1 mM EDTA
2X Binding Buffer
40 mM Tris-HCl, pH 7.6
1 M NaCl
2 mM EDTA
to make 100 mls:
0.2 g SDS
0.4 ml 0.5 M EDTA
5.84 g NaCl
dissolve in 96 ml final dH2O. Add 0.1 ml DEP. Autoclave.
When cool add 4.0 ml 1 M RNAse free Tris-HCl, pH 7.6.
10 mM Tris-HCl, pH 7.6
1 mM EDTA
to make 500 ml:
1.0 ml 0.5 M EDTA