Laboratory 1. genomic DNA Extraction and Gel Electrophoresis Exercise 1. DNA Extraction using CTAB: Materials: CTAB solution β-mercaptoethanol Liquid nitrogen 3M NaOAc Phenol/EB pH 8.0 100% isopropanol 96:4 (chloroform: Isoamyl) 70% EtOH Elution Buffer (EB) 1. Pre heat 500μl CTAB in 65ºC water bath. 2. Place 0.2 to 0.5g plant material into the mortar and add liquid nitrogen, grind to a powder. 3. Add plant material to the pre-heated CTAB and add1%β-mercaptoethanol (5μl) to the centrifuge tube and incubate at 65˚C for 0.5 hr. Vortex well at least every 10min. 4. Centrifuge for 8min. at 13,000rpm, transfer top layer to new microcentrifuge tube. 5. Add 500ul 96:4 chlorofor:isoamyl alcohol, vortex well, centrifuge for 3min., transfer top layer to a new microcentrifuge tube. 6. Repeat step 5. 7. Add 0.1 volume 3M NaOAc and 0.7 volumes 100% isopropanol, vortex, incubate in ice bath for 10min. Centrifuge for 10min. at 13,000 and discard supernatant. Be careful not to disturb the pellet. 8. Wash pellet in 500μl cold 70% EtOH, invert 5X; incubate 10min. in an ice bath. 9. Centrifuge for 2min. add maximum speed to secure the pellet, discard the supernatant. 10. Dry the pellet by placing inverted, cap open, in the 37o incubator for 10 min. 11. Add 50μl EB Buffer, store in 4˚C overnight before quantifying. CTAB Solution: 2g CTAB 2g PVP 28ml 5M NaCl (29.22g/100ml) 4ml EDTA pH 8.0 10ml 100mM TrisHCl pH8.0 1% β-mercaptoethanol (just before use) ·Prepare the solution without CTAB, PVP, β-mercaptoethanol and autoclave for 20 min. ·After autoclave add CTAB and PVP; heat to dissolve CTAB and PVP on the hot tray. Add the 1% β-mercaptoethanol directly to the extraction vial (5μl/500μl of CTAB solution). Pre-heat the solution in a 65˚C water bath before use. General Considerations and Rationale Three components are necessary for successful nucleic acid extraction: 1) inhibition of nucleases, 2) removal of proteins, and 3) physical separation of the nucleic acid from other cellular components. Nuclease inhibition and removal of proteins are not mutually exclusive and often are accomplished in a single step (such as phenol extraction). Inhibition of Nucleases Detergents inhibit nucleases and help separate the proteins from the nucleic acids. A common detergent is sodium dodecylsulfate (SDS). Avoid the use of potassium salts or temperatures below 10 °C with SDS since this may cause precipitation of the detergent. One of the most widely used detergents for plant DNA extraction buffers is CTAB (cetyltrimethyl-ammonium bromide). CTAB is most often used in a 2% (w/v) solution (Rogers & Bendich 1985, Doyle & Doyle 1987, Nickrent 1994). Removal of Proteins Phenol is a strong denaturing agent for proteins. In phenol extractions, proteins partition into the organic phase (and interface) whereas nucleic acids partition in the aqueous phase. Usually phenol is used in a 1: 1 mixture with chloroform since deproteinization is more effective when two different organic solvents are used simultaneously. In addition to denaturing proteins, chloroform is useful in removing lipids and a final chloroform extraction helps to remove the last traces of phenol. The isoamyl alcohol helps with the phase separation, decreases the amount of material found at the aqueous and organic interface, and helps reduce foaming. Antioxidants such as 8-hydroxyquinoline or ß-mercaptoethanol are often added to phenol. During phenol extractions, the pH of the buffer is important in determining whether DNA and/or RNA are recovered. At pH 5-6, DNA is selectively retained in the organic phase leaving RNA in the aqueous phase (hence water saturated phenol is useful for RNA extractions). At pH 8.0 or higher, both DNA and RNA are retained in the aqueous phase. Phenol can be stored under buffer for up to one month. Protease The most common proteolytic enzyme used in nucleic acid extractions is protease K (e.g. Sigma P4755) which is generally made up in 50 units/ml aliquots and stored frozen for immediate use during extractions. Solutions are generally incubated with this enzyme at 37 °C for at least one hour. Other Extraction Buffer Components Two common reducing agents found in extraction buffers are ß-mercaptoethanol or dithiothreitol (DTT). EDTA is also present to chelate Mg+2 ions thus mediating aggregation of nucleic acids to each other and to proteins. Ethanol and/or Isopropanol Precipitation The most common method of concentrating nucleic acids is with alcohol precipitation. This occurs in the presence of a salt (see below) at low temperatures (-20 °C or less). Either 2.0 volumes of ethanol can be used to precipitate DNA or 0.6 volumes of isopropanol. Isopropanol is often used for the first precipitation, but not for final ones because it tends to bring down salts more readily than ethanol. Isopropanol can also be used when the volume of the tube will not allow the addition of 2 volumes of ethanol. The nucleic acid is collected by centrifugation at 10,000 rpm for ca. 20 - 30 minutes. The most common monovalent cations used in nucleic acid precipitations are shown in the following table: Stock Solution Final Concentration Sodium acetate 2.5 M (pH 5.2 - 5.5) 0.25 M to 0.3 M 0.10 M (optimal < 0. 15 M = 1/50 Sodium chloride 5.0 M vol.) Ammonium 4.0 M 1.3 M acetate* * Filter sterilize, do not autoclave The choice of salt is determined by the nature of the sample and the intended use of the nucleic acid. Samples with phosphate or greater than 10 mM EDTA should not be ethanol precipitated because the salts will come down with the nucleic acid. Butanol Extractions DNA is recovered from dilute solutions by extracting with 2-butanol. The water from the sample moves into the butanol which is discarded, thus leaving a higher DNA concentration in the aqueous phase. Water saturated butanol is also used to remove residual ethidium bromide from samples obtained via CsCl centrifugation or agarose gel purification. Reagents: 2X CTAB buffer: 100 mM Tris-HCI, 1.4 M NaCl, 30 mM EDTA, and 2 % (w/v) CTAB. The buffer is usually ca. pH 8.0 without adjustment. protease K (Sigma Type H, ca. 0. 13 units/mg, P-4755). I store the enzyme in 50 units/1 ml aliquots in the freezer. Dithiothreitol (DTT) 0.5 M. Stored as 0.5 ml aliquots - added to buffer just before use. 1X TE buffer Ammonium acetate, 4.0 M. Organics: chloroform:isoamyl alcohol (24: 1), Tris buffered phenol, cold isopropanol, cold 100% ethanol, and 70% ethanol. The best DNA extracts come from young plant material. Attempt to conduct the extraction soon after collection if refrigeration is not possible. Day 2 Exercise 2. Gel Electrophoresis The standard method for separating DNA fragments is electrophoresis through agarose gels. DNA is applied to a slab of gelled agarose and then an electric current is applied across the gel. Because DNA is negatively charged (phosphate groups in the backbone), it migrates through the gel towards the positive electrode. The rate of migration depends on: 1) the size of the fragment; the smaller the DNA, the faster it "runs" through the gel; 2) the concentration of agarose; the higher the concentration, the more the agarose retards the movement of the DNA fragments; and 3) the voltage applied to the gel; the higher the voltage the quicker the DNA "runs" (but at a trade-off--the DNA fragments do not separate as efficiently). a. Pouring the gel A demonstration on preparing and running an agarose gel will be given. Each group should have a gel apparatus and cover, two end walls, two combs and a plastic gel tray. The gel tray should be placed in the chamber and the two end walls inserted. A comb should be placed near one of the end walls. Make 100 ml of a 1.0% agarose in 1X TBE for your gel in a 250 ml flask. Minigels require ~30 ml agarose. Use the microwave to melt the agarose. Be sure the solution is completely melted and homogenous. Warning!! Never leave the agarose solution unattended when using the microwave. The solution must be frequently swirled during the heating process to prevent superheating of local areas. Always use "hot hands" or autoclave gloves when heating the agarose. You will be provided Ethidium Bromide (EtBr) at a concentration of 1 mg/ml in a 1.5 microfuge tube. This tube should be saved and stored at 4ºC. Warning!! Ethidium bromide is a mutagen. Remember to wear gloves when working with EtBr and to dispose of contaminated tips in specially marked containers. Gels should be placed in a plastic bag for disposal. Microwaving EtBr produces harmful volatiles. Add EtBr to a final concentration of 0.1 to 0.5 g/ml to agarose immediately before you pour the gel. To do this, label a 50 ml conical tube EtBr/agarose. Add the EtBr to the tube and then pour about 30 mls of the agarose into the tube. Cap and invert a few times. The agarose solution should be cooled to about 50ºC (a temperature at which one can hold the flask, but it is uncomfortable) before pouring the gel. Pouring the gel when the agarose is too hot may damage the gel apparatus. Pour the 1.0% agarose minigel with 0.1 g/ml EtBr and allow it to solidify. Practice loading a few lanes with loading dye if you've never loaded a gel before. This can be run into the gel without affecting the migration of DNA added to the well at a later time. At first it will be easier to load the wells dry. Add 1X TBE buffer to submerge the gel prior to electrophoresis. Later, you should become adept at loading wells submerged in buffer. b. Loading the Samples and Running the Gel: To a new eppendorf or on parafilm, add 5 l loading dye to 15 l of your sample and finger flick. Load 20 l total volume of your samples per well for minigels with 8-well combs. Load 10 l of the molecular weight markers provided. Your gel should contain: 1. molecular weight markers 2. gDNA group 1 3. gDNA group 2 4. gDNA group 3 Run the gel at 100 V until the bromophenol blue is about 3/4 through the gel (approx. 1 h). Using gloves, carefully remove the tray with the agarose gel. Take it to the UV transilluminator and slide the gel off the tray onto the lamp. Wearing UV-protective goggles (NOT YOUR SAFETY GLASSES), examine the gel. The cover of the transilluminator may also be used to protect your eyes. Photograph your gel with a ruler for your notebook. Exercise 3. Quantifying DNA Using a Bio Photometer (Spectrophotometer) (may be done at the second class) - turn the BioPhotometer on and allow to warm up (switch on back left) - choose the correct wavelength by pressing the appropriate button (dsDNA) - Put 49 l of EB buffer into a clean cuvette (be careful to touch only the frosted sides of the cuvette) - Insert the cuvette into the port and “blank” the machine - Remove the cuvette and add 1 l of you DNA - Record the readings BioPhotometer Display Readout A) g/ml B) 260/280 Detection of DNA and RNA with Absorption Spectroscopy. C) 260/230 A - This number gives you the concentration of nucleic acids (DNA and RNA) measured as micrograms per milliliter (g/ml) Values B and C give you an idea of how clean your sample is. B - The ratio of the absorbencies of the sample at two discreet wavelengths of light – 260 nanometers (nm) and 280 nm. Nucleic acids have a peak absorption at 260 nanometers while proteins have a peak absorption at 280 nm. Protein contamination will reduce the 260/280 ratios. C - The ratio of the absorbencies of the sample at two discreet wavelengths of light – 260 nanometers (nm) and 230 nm. Again, nucleic acids have peak absorption at 260 nanometers while contaminants containing peptide bonds and phenols have peak absorption at 230 nm. Relatively higher contaminant concentrations will reduce the 260/230 ratios.
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