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# Chapter 3: Measurement of DNA Concentration Before further analysis, it is important to determine the concentration and condition of the DNA that you have isolated. This can be done in three ways: by examining UV absorbance with a spectrophotometer; by fluorimetry; or by comparison with DNA stan...

# Chapter 3: Measurement of DNA Concentration Before further analysis, it is important to determine the concentration and condition of the DNA that you have isolated. This can be done in three ways: by examining UV absorbance with a spectrophotometer; by fluorimetry; or by comparison with DNA standards on concentration gels. Using a spectrophotometer, the amount of ultraviolet radiation absorbed by a solution of DNA is directly proportional to the amount of DNA in the sample. The concentration of DNA in a sample can be determined given that a 50 µg/mL solution of double-stranded DNA (ds DNA) has an absorbance of 1.0 at 260 nm. Single-stranded DNA (ss DNA) and RNA in concentrations of 40 µg/mL have absorbances of 1.0 at 260 nm. The purity of a DNA preparation can be judged by examining the ratio of absorbances at 260 nm and 280 nm. Pure DNA and RNA have 260/280 absorbance ratios of 1.8 and 2.0, respectively; protein or phenol contamination will lower the absorbance ratios (and result in reduced accuracy of DNA quantitation and poor restriction digestion). DNA prepared by some protocols may contain a large amount of RNA and pigments that can cause spuriously high estimation of DNA concentration on a spectrophotometer. For this reason, 0.8% agarose concentration gels are useful for assessing both the quantity and quality of the genomic DNA (is it high molecular weight, or is there substantial shearing or degradation?) and the amount of RNA present. The use of a low percentage agarose gel (0.4%) allows separation of nuclear and mitochondrial DNA for vertebrates and most invertebrates, potentially allowing the assessment of the proportion of nuclear versus mitochondrial DNA. ## Spectophotometric Determination of DNA Concentration 1. Turn on spectrophotometer UV-lamp and after it has warmed up, set the wavelength at 260 nm. 2. Determine the volume of DNA to use by estimating concentration. Most genomic DNA preparations yield DNA concentration of about 100 µg/mL while plasmid DNA preparat will often result in a DNA concentration of 1 mg/mL. An absorbance (optical density or OD) of 0.05 to 0.1 is high enough for an accurate assessment. Thus the sample volume generally ranges from 5 to 50 µL. 3. While the machine is warming up, Prepare test sample by taking the appropriate volume of each sample and adding TE to a final volume of 500 µL in a microfuge tube or microtitre plate well. Uniform mixing of the sample is essential. 4. Fill one cuvette (the “Zero” or blank cuvette) with 500 µL TE and place in spectopgtometer at 260 nm. “Zero” the machine or note the reading. (The same Zero setting should be sufficient for both 260 and 280 nm readings, but check.) 5. Fill the other cuvette (the test cuvette) with 500 µL TE and take readings at both 260 and 280 nm. Any differences between the cuvettes need to be noted so adjustments to the sample reading can be made. 6. Rinse out the test cuvette with sterile water and tamp out on a paper wipe. Fill the test cuvette with 500 µL from the test sample. Take absorbance readings at 260 nm. Then again at 280 nm. 7. Rinse out cuvette with sterile water, tamp dry and repeat step 6 for each sample. It is often wise to occasionally check the “Zero” setting with blank cuvette. 8. Taking into account the initial dilution. The sample concentration is calculated as: DNA conc. (µg/mL) = (500 µL/x µL) (50 µg/mL) (OD260) where x is the volume of DNA used. **Note:** a-1 O.D.260= 50 µg of ds DNA /mL = 50 µg / 1000 µL = 0.05 µg / µL # Chapter 4: DNA Analysis by Gel Electrophoresis ## Introduction Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and proteins. Agarose is a polysaccharide derivative of agarose. The agarose gel contains microscopic pores which act as a molecular sieve. The sieving properties of the gel influences the rate at which a molecule migrates. Smaller molecules move through the pores more easily than larger ones. Molecules can have the same molecular weight and charge but different shapes. Molecules with a more compact shape (a sphere is more compact than a rod) can move more easily through the pores. Agarose gel electrophoresis possesses a great resolving power, yet is relatively simple and straightforward to perform. The gel is made by dissolving agarose powder in boiling buffer solution. The solution is then cooled to approximately 50°C and poured into a mold where it solidifies. The gel is submerged in a buffer-filled chamber which contains electrodes. DNA samples are prepared for electrophoresis by mixing them with solutions containing glycerol or sucrose. This makes the samples denser than the electrophoresis buffer. These samples can then be loaded with a micro-pipet or transfer pipet into wells that were created in the gel by a comb during casting: the dense samples sink through the buffer and remain in the wells. A direct power supply is connected to the electrophoresis apparatus and current is applied. Charged molecules in the sample enter the gel through the walls of the wells. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). ## CTAB Method: Remove Polysaccharides Followed By Protein Removal and DNA Separation **CTAB Lysis Buffer:** Used to liberate and complex with total cellular nucleic acids. **EDTA:** Chelates Mg 2+ to inactivate nucleases, preventing DNA degradation during extraction. **Noel:** Helps to remove proteins that are bound to DNA, keep protein dissolved in aqueous layer. **Cold Iso Prop:** Prevent dissolution of DNA (DNA is precipitated by cold acidic buffer) DNA pH is sensitive during cell lysis. **Tris:** Stabilize DNA pH. **PVP:** Reduce the effect of phenolic poly during extraction. **CTAB:** Disrupt membrane. **Chloroform Isoamyl:** Separate contaminants into organic phase and nucleic acid in aqueous phase. **Isopropanol:** Allows ppt. of DNA. **Ethanol 70%:** During washing steps allows the salts to dissolve while minimizing DNA solubility. **TE (Tris-edta):** Dissolve DNA ppt # Buffers of Gel Electrophoresis * **TAE:** Tris acetate buffer (50x stock) * **TBE:** Tris - phosphate buffer (50x stock) * **TPE:** Tris - borate buffer (50x stock) * **Alkaline buffer:** Used to separate plasmid (circular DNA of bacteria) - separate 3 bands on 0.8% agarose gel. **Bromophenol Blue:** Separate 200-400 bp DNA (small molecule) **Xylene Cyanol:** 4kb DNA (large molecule) **Loading dye:** *Glycerol or sucrose or ficoll to dense. *Bromophenol blue or xylene cyanol or twice to give colors. **Ethidium bromide:** Staining of DNA. 1000x stock soln. 0.5 mg/mL. # Absorbance of RNA 40 ug/mL at 260 nm = 1.0 * 0.D260 = 40 µg of RNA or ss DNA /mL = 40 µg / 1000 mL = 0.04 µg / µL. Conc. of ss DNA or RNA µg/µL = 0.1D260 x 0.04 x dilution factor # DNA Analysis by Gel Electrophoresis Agarose gel electrophoresis is a method used to separate molecules according to charge, size and shape. Agarose is a polysaccharide derivative of agarose: it contains pores (act sieve or network). The smaller molecules move through pores easier than the large one. DNA samples are prepared for electrophoresis by mixing them with loading dye containing glycerol or sucrose to denoise the samples more than the tank buffer. Samples must be loaded into the wells, then a direct power supply is connected. Charged molecules of DNA having a -ve charge migrate to the the electrode anode. The buffer which exist in the cell act as a conductor of electricity of controlling pH. pH is important to the charge of stability of molecules. # After Migration of Molecules DNA Must Be Stained By Using (Ethidium Bromide) This compound makes a complex with the DNA so it can be detected by UV transilluminator. ## Preparation of Gel (Agarose Gel Prep.) - **If Genomic DNA:** - 0.8 g agarose - 2 mL TAE buffer (50x) - 98 mL H2O - Heat on hot plate until give clear solution. - Leave to cool. - Add 100 µL ethidium bromide (1000x). - Mix well. Then pour into the cell electrophoresis where the agarose solidifies. Then submerge the gel in a buffer filled chamber containing electrodes. - **If: PCR product:** - 1.5 - 2 g agarose - 2 mL TAE (50x) - 18 mL H2O - Heat on hot plate until give clear solution. - Leave to cool. - Add 100 µL ethidium bromide (1000x). - Mix well. Then pour into the cell electrophoresis where the agarose solidifies. Then submerge the gel in a buffer filled chamber containing electrodes. **Note:** The tank buffer is a TAE buffer also (50x). The cell takes 1500 mL buffer. # Agarose Gel Electrophoresis Of DNA ## Materials and reagents * An electrophoresis chamber and power supply. * Gel casting trays: Available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. * Sample combs: Molten agarose is poured around these to form sample wells in the gel. * Electrophoresis buffer: Usually Tris-acetate-EDTA = TAE) or Tris-borate-EDTA (TBE). (For preparation of 1L of 50X TAE: 242g Tris base + 57.1mL glacial acetic acid + 100mL of 0.5M EDTA, then adjust the pH at 8) * Loading buffer: Contains something dense e.g. glycerol to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded (Composition of 6X loading dye: 10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA). * Ethidium bromide: A fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical. Use gloves while handling. * Transilluminator: An ultraviolet lightbox, which is used to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light. ## Procedure 1. To prepare 100 mL of a 0.7% agarose solution, measure 0.7 g agarose into a glass bottle and add 100 mL 1X TAE buffer. Microwave or stir on a hot plate until agarose is dissolved and solution is clear. 2. Allow solution to cool to about 55°C before pouring (Ethidium bromide can be added at this point to a concentration of 0.5 µg/mL) 3. Prepare gel tray by sealing ends with tape, place comb in gel tray about 1 cm from one end of the tray and position the comb vertically such that the teeth are about 1-2 mm above the surface of the tray. 4. Pour 50°C gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature. 5. To run, gently remove the comb and tape, place tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the gel). Excess agarose can be stored at room temperature and remelted in a microwave. To prepare samples for electrophoresis, add 1 µL of 6x gel loading dye for every 5 µL of DNA solution. Mix well. Load 5-12 µL of DNA per well (for mingei). Electrophorese at 50-150 volts until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized. 6. If the gel was not stained with ethidium during the run, stain the gel in 0.5 µg/mL ethidium bromide until the DNA has taken up the dye and is visible under short wave UV light, if the DNA will not be used further, or with a hand-held long-wave UV light if the DNA is to be cut out and purified. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis. # Migration of DNA Fragments in Agarose 1. Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear. 2. Several additional factors have important effects on the mobility of DNA fragments in agarose gels. Chief among these factors are: * **Agarose Concentration:** By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilities separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. The larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. * **Voltage:** As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel) * **Electrophoresis Buffer:** Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. # 2.1.4 Buffers ## Materials and methods: * **Agarose gel-loading buffer 6X stock:** - Bromophenol Blue: 50 mg (0.25% final) - Sucrose: 8 gm (40% final) - Distilled water: complete to 20 ml * **Alkaline phosphatase buffer (AP buffer) for western blot:** - 1 M Tris-HCl pH 9.5: 10 ml (0.1 M final) - 5 M NaCl: 2 ml (0.1 M final) - 1 M MgCl2: 0.5ml (5 mM final) - Distilled water: complete to 100 ml * **Citrate / Saline buffer:** - NaCl: 8.5 gm - Na citrate: 15 gm - Distilled water: complete to 1 liter * **Fusion protein extraction buffer:** - 1 M Tris-HCl pH 8.0: 50 ml (50 Mm final) - 5 M NaCl: 30 ml (0.15 M final) - 0.5 M EDTA pH 8.0: 10 ml (1% final) - Distilled water: complete to 1 liter - 100 mM PMSF freshly added to the solution to 1mM final concentration. # STE buffer * 10 mM Tris-Cl, pH 7.5 * 10 mM NaCl * 1 mM EDTA # STET solution * 8% sucrose * 0.5% Triton X-100 * 50 mM EDTA * 50 mM Tris-Cl, pH 8 # Storage buffer * 10 mM Tris-Cl, pH 8 * 10 mM NaEDTA, PH 8 # Stop solution * 10 mM Tris-Cl, pH 7.5 * 20% glycerol * 0.1% SDS * 0.1% Bromphenol Blue # Sucrose solutions, 10% and 40% * 10%-sucrose or 40% sucrose * 1 M NaCl * 20 mM Tris-Cl, pH 7.5 * 5 mM EDTA # Sucrose/Tris/EDTA solution * 25% sucrose * 50 mM Tris-Cl, pH 8 * 100 mM EDTA # Suspension medium (SM) * 5.8 g NaCl * 2 g MgSO4.7H2O * 50 ml 1 M Tris-Cl, pH 7.5 * H₂O to 1 liter * Solid gelatin (Difco) to 0.1% * Heat to just below boiling to dissolve # Suspension TBS (STBS) solution * 25 mM Tris-Cl, pH 7.4 * 137 mM NaCl * 5 mM KCI * 0.6 mM Na2HPO4 * 0.7 mM CaCl₂ * 0.5 mM MgCl2 * Make up in distilled H₂O and filter sterilize. A 10x stock of this solution can be made that is diluted to 1x with sterile distilled H₂O prior to use. # T4 DNA ligase buffer, 2x * 100 mM Tris-Cl, pH 7.5 * 20 mM MgCl2 * 20 mM DTT # TA buffer, 5x * 200 µl 1 M Tris-acetate, pH 7.8 * 400 µl 1 M potassium acetate * 60 µl 1 M magnesium acetate * 3 µl 1 MDTT * 105 µl 5 mg/ml BSA * 432 µl H₂O # TAE electrophoresis buffer * **50x stock solution, pH-8.5:** - 242 g Tris base - 57.1 ml glacial acetic acid - 37.2 g Na₂EDTA-2H₂O - H₂O to 1 liter # 250 Taq DNA polymerase * Taq polymerase is usually supplied at 5 U/µl. Most templates have an optimum from 1.5 to 2.5 U/100 µl reaction. Immediately before use dilute into cold 1x PCR reaction buffer to give the desired enzyme concentration. # TBE electrophoresis buffer * **10x stock solution:** - 108 g Tris base - 55 g boric acid - 40 ml 0.5 M EDTA, pH 8.0 - H₂O to 1 liter * **1x working solution:** - 89 mM Tris base - 89 mM boric acid - 2 mM EDTA