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1 MOLECULAR BIOLOGY LABORATORY SCHEDULE – TENTATIVE SPRING SEMESTER 2007 PCB4524L—Tues., Wed., or Thurs. 2:30-5:15pm Building 58; Room 144 Week of: 1/22 1/29 2/5 2/12 2/19 2/26 Introduction to Molecular Biology Lab Isolation of Total RNA and DNA from Whole Cells – Part I Isolation of Total RNA and DNA from Whole Cells – Part II Determining the Concentration and Purity of DNA and RNA in Solution Restriction Endonuclease Digestion of λ DNA with Hind III, Run gel-Transformation of Competent E. coli JM109 Cells with Recombinant Plasmid Containing the Gene for Green Fluorescent Protein--Part I Southern Blot LAB EXAM ONE 3/12 3/19 3/26 4/2 PCR and DNA Fingerprinting –Part I Spring Break—Have a safe Holiday!! PCR and DNA Fingerprinting –Part II Restriction Endonuclease Digestion of pGEM3Z Plasmid Vector with Hind III and Ligation.of λ DNA Fragment into Plasmid. Blue/White Colony Selection 3/5 4/9 4/16 4/23 LAB FINAL EXAM 2 LAB REPORT GUIDELINES FOR MOLECULAR BIOLOGY 1. 2. Reports are due 1 week after the completion of a lab experiment. Reports must be word processed using a font size of 12 pt and DOUBLE SPACED. 3. All reports will contain the following elements in the following order. a. Title Page (name of experiment, your name, names of lab partners, date) b. c. d. Introduction – state the purpose of the experiment Methods – ONLY include modifications made to the written protocol Results – concisely state the results obtained, show data in the form of tables and/or figures. e. Discussion/Conclusions – state what conclusions you can make based on the data you obtained f. A copy of the lab protocol 4. 5. 6. Grading is based on a 10 pt scale. Format, content, accuracy, spelling, and grammar will be accessed. Failure to follow the prescribed format will result in an automatic 2 pt deduction. 7. Late submissions will result in an automatic 2 pt deduction. Submissions that are overdue by 1 week or more will receive a grade of ZERO. 3 Molecular Biology Laboratory 1 Isolation of Total RNA and DNA from Whole Cells or Tissues. Part One Introduction and Background A fundamental technique in molecular biology is the isolation of RNA and/or DNA from prokaryotic and eukaryotic cells. RNA samples must be intact and essentially free of contaminating DNA and protein. The total RNA sample can then be used for Northern blotting, isolation of poly(A) mRNA, in vitro translation, RNase protection assays, cloning, and polymerase chain reactions (PCR). DNA samples can be used for PCR, digestion with restriction enzymes, and Southern blotting et cetera. The method employed in this laboratory will be a modification of the procedure developed by Chomczynski and Sacchi (1). It employs a single step liquid phase separation that results in the simultaneous isolation of RNA, DNA, and protein (2). A monophase solution of guanidine thiocyanate and phenol sold under the name of TRI REAGENT TM is used. TRI REAGENT dissolves DNA, RNA, and protein when cells are lysed. When chloroform is added to the homogenate the mixture separates into 3 phases: an aqueous phase containing RNA, the interface containing DNA, and an organic phase containing protein. Each ml of TRI REAGENT is sufficient to isolate RNA, DNA, and protein from 50 – 100 mg of tissue, 5 – 10 x 106 cells, or 10 cm2 of surface area of tissue culture cells in a monolayer. Once cells have been lysed in TRI REAGENT, the samples can be stored at -70 oC for up to 1 month. Reagents required for: RNA Isolation Chloroform Isopropanol 75% EtOH 0.5% SDS solution or DEPC-treated water DNA Isolation 8 mM NaOH 0.1 M sodium citrate/10% EtOH Absolute EtOH Protein Isolation Isopropanol Absolute EtOH 95% EtOH 1% SDS 0.3 M guanidine hydrochloride in 95% EtOH 4 NOTE: Wear gloves when performing all procedures. This not only protects you from the harsh chemicals used, but prevents exposure of samples to the RNase present on your skin. Even a tiny amount of RNase contamination is sufficient to severely degrade RNA. All pipet tips, microfuge tubes, electrophoresis buffer tanks, reagents and any other item that comes into contact with RNA samples should be made RNase-free by appropriate means (autoclaving, treatment with diethylpyrocarbonate or other RNase inhibitor). PROTOCOLS I. Sample Preparation 1. Obtain a logarithimic phase culture of Tetrahymena thermophila and pipet 1.5 ml of cell suspension into a sterile 2 ml microfuge tube. 2. Sediment the cells by centrifuging for 3 min at the maximum setting on the microcentrifuge. A firm cell pellet should result. If not, repeat the centrifugation. 3. Decant the supernatant (growth medium) into a waste container. Using a P-200 pipet, aspirate any residual medium from the surface of the pellet being careful not to remove any cells. 4. Add 1 ml of TRI REAGENT to the cell pellet, and lyse the cells by repeated pipeting using a P-1000 pipet. 5. Let the sample stand for 5 min at room temperature to allow for the complete dissociation of nucleoprotein complexes. 6. Add 200 μl chloroform. Cap the microfuge tube and shake vigorously for 15 sec. 7. Allow the sample to stand for 2 to 15 min at room temperature, then centrifuge for 15 min at 12,000 x g. 8. Following centrifugation the sample mixture should separate into 3 phases: 1. A colorless, upper aqueous phase containing total RNA. 2. An interface containing DNA. 3. A red, lower organic phase containing protein. 5 II. Isolation of Total RNA 1. Transfer the upper, aqueous phase to a fresh, sterile microfuge tube and add 500 μl isopropanol to precipitate the RNA. Store the remaining sample (interface plus lower phase) tightly capped at 4 oC. Allow the RNA sample to stand for 5 – 10 min at room temperature. 2. 3. Centrifuge the sample for 10 min at 12,000 x g. The RNA will be present as a pellet on the side and bottom of the tube. Using a pipet, remove the supernatant to a waste container and wash the RNA pellet by adding 1 ml of 75% ethanol. 4. 5. Vortex the sample to resuspend the pellet, then centrifuge at 12,000 x g for 5 min. Using a pipet, remove the supernatant from the RNA pellet, then add 100 μl of fresh 75% ethanol. Cap and label the tube. Store at -20 oC. 6. 7. (1) Chomczynski, P. and Sacchi, N. 1987. Anal. Biochem., 162, 156-159. (2) Chomczynski, P. 1993. BioTechniques, 15, 532-537. 6 Molecular Biology Laboratory 2 Isolation of Total RNA and DNA from Whole Cells or Tissues Part Two Remove the RNA samples stored in 75% ethanol from the freezer. II. Isolation of Total RNA (continued) 1. Centrifuge the RNA sample for 10 min at 12,000 x g. 2. Remove ALL of the alcoholic supernatant from the pellet, using a pipet. 3. Air-dry the pellet for 5-10 min. Do NOT allow the RNA pellet to dry completely. This will greatly decrease its subsequent solubility! 4. Add 10 μl of Rnase-free water to the pellet. 5. Solubilize the RNA by repeatedly heating briefly at 55 oC and pipeting with a P200 micropipet. This may take 10 to 15 min. 6. Store the solubilized RNA sample at -20 oC. III. Isolation of DNA Remove the tubes containing the lower phase and interface from the refrigerator. 1. Carefully remove all of the remaining aqueous phase overlaying the interface and discard in a waste containing. It is critically important to remove ALL of the remaining aqueous phase prior to DNA precipitation to ensure high quality DNA. 2. Add 300 μl of 100% ethanol to the sample and mix by inversion 2 or 3 times. 3. Let the sample stand for 2-3 min at room temperature to precipitate the DNA. 4. Centrifuge at 2,000 x g for 5 min. 7 5. Remove the supernatant from the DNA pellet and discard. (Note: IF the supernatant were saved, protein could be isolated from it.) 6. Wash the DNA pellet by adding 1 ml of 0.1 M sodium citrate/10% ethanol solution. Allow the sample to stand for 30 min with occasional mixing by pipet. Do NOT reduce the time the DNA is in the wash solution. This step removes phenol from the sample and 30 min is the ABSOLUTE MINIMUM time required. 7. Centrifuge the sample for 5 min at 2,000 x g, then remove the supernatant. 8. Repeat the washing step as described in 6 above. 9. After centrifugation and removal of the second wash solution, resuspend the DNA pellet in 1.5 ml 75% ethanol and allow to stand for 10 min at room temperature. 10. Centrifuge at 2,000 x g for 5 min. Remove the supernatant by pipet. 11. Air-dry the pellet for 10 min. 12. Solubilize the DNA by adding 100 μl of 8 mM NaOH with repeated SLOW pipeting with a P-200. Mixing to aid dissolution of the DNA pellet MUST be done slowly and gently as high molecular weight DNA is susceptible to breakage by mechanical shearing! The mild alkaline solution greatly improves the solubility of the DNA. 13. Centrifuge the solubilized DNA at 12,000 x g for 10 min to remove any insoluble material. Carefully transfer the supernatant to a clean, sterile microfuge tube and discard the pellet. A viscous supernatant indicates the presence of high molecular weight DNA. 14. Samples stored in 8 mM NaOH are only stable for approximately 24 h due to the elevated pH. Add 7.5 μl of 0.1 M HEPES (free acid) to adjust the pH to between 7 and 8, and add 1 μl of 100 mM EDTA per 0.1 ml of DNA solution. Mix by inversion 2 or 3 times. 15. Store the DNA sample at -20 oC. 8 Molecular Biology Laboratory 3 Determining the Concentration and Purity of DNA and RNA in Solution. Introduction and Background Nucleic acids, like many other substances, have the property of absorbing light at a specific wavelength. DNA and RNA absorb light maximally at a wavelength of 260 nm. Because of this property, they can be quantified spectrophotometrically using the BeerLambert equation: A = εcl where A = absorbance, ε = the molar extinction coefficient, c = the molar concentration of the molecule in solution, and l = the length of the light path through the solution. Molecular biologists usually quantify nucleic acids in absorbance units (AU) or optical density units (OD). One AU or OD unit of nucleic acid is defined as the concentration that gives 1 AU at a given wavelength. The quantitative basis for this definition is obtained by rearranging the Beer-Lambert equation: OD = εcl C 1 ___ = ___ = 1 AU OD ε Therefore, C / OD defines 1 AU. 1 AU is the inverse of the absorption (extinction) coefficient. To calculate the concentration of nucleic acid: C = 1 AU x OD x DF, where DF is the dilution factor and concentration is expressed in terms of mg / ml. The following table gives absorption constants for important nucleic acids. Nucleic Acid ε1mg/ml 1 AU260 nm 1cm (μg/ml) ___________ _____ _________ 9 dsDNA ssDNA RNA ss oligonucleotide(a) 20 25 25 ≈30 50 40 40 ≈33 ss oligonucleotide(b) ≈40 ≈25 ___________________________________________________ (a) (b) 60 – 100 nucleotides long less than 40 nucleotides long The purity of a nucleic acid solution can be determined by calculating the A260/A280 ratio. The nucleic acid absorbs maximally at 260 nm and protein (a principle contaminant) absorbs maximally at 280 nm. Pure DNA has an A260/A280 ratio of 1.8. Pure RNA has an A260/A280 ratio of 2.0. If the ratios are significantly less than these values, then the sample is not pure. The following page shows sample calculations for determining the concentration of a DNA sample by using (1) the absorption (extinction) coefficient (expressed as mg/ml) or (2) by using the value of 1 AU in μg/ml. Data for the sample calculation: 10 μl of a solution of dsDNA is diluted with 1.99 ml of water and placed in a cuvette. The absorbance of the diluted sample is determined at 260 nm and found to be 0.50. What is the concentration of the diluted DNA in the cuvette? What is the concentration of the undiluted (original) sample of DNA? 10 Method (1) Method (2) …most commonly used Since the result of interest is always the CONCENTRATION OF DNA IN THE ORIGINAL SAMPLE, the only equation needed is C = 1 AU x OD x DF. It also represents the SIMPLEST way to calculate the concentration. Since we are doing some calculations…. it is usually necessary to add a controlled amount of oligonucleotide primer in PCR reactions, and for other purposes. Typically, the amount of ss oligonucleotide primer required is a specified number of pmol, therefore, it would be advantageous to review how to calculate the total amount of ss DNA in terms of pmol. The following page gives an example of such a calculation. 11 You emailed a colleague and requested that she send you a small amount of primer DNA for you to use in a PCR reaction. Your colleague, in the traditional spirit of science, sent you the primer you requested. You open the package and find a microfuge tube with a label on it. From the label you learn that the microfuge tube contains: 200 μl of the 20-mer (a 20 base ss DNA primer) and that the solution has an OD of 0.45 at 260 nm. Calculate the following: 1. the concentration of the primer solution in μg/ml 2. the μM concentration of the primer 3. the total number of pmol in the primer sample Solutions: 1. C = 1 AU x OD x DF C = (25 μg/ml) (0.45) (1) C = 11.2 μg DNA / ml For the next calculation, you need to know that the approximate mass of 1 base is 325 daltons (D). Your sample is a 20-mer, it has 20 bases, so: (325 D / base) x (20 bases) = 6,500 D = 6,500 g/mol 2. M wt MW L 1.12 x 10-5 g ________________________ (6,500 g/mol) (2 x 10-4 L) = = 8.65 x 10-6 mol/L = 8.65 x 10-6 mol/L x (1 μM / 1 x 10-6 mol/L) = 8.65 μM 12 3. M = mol/L rearrange to give, mol = M x L = (8.65 x 10-6 mol/L) (2 x 10-4 L) = 1.73 x 10-9 mol = 1.73 x 10-9 mol x (1 pmol / 1 x 10-12 mol) = 1,730 pmol OK. Enough calculations for now. Here‘s information on the effect of temperature on DNA melting and reannealing. In intact, ds DNA, the bases are paired and stacked. The result is that absorbance is decreased. When ds DNA solutions are heated to a sufficiently high temperature, the hydrogen bonds holding the complimentary bases together begin to break. This does not occur all at once. GC pairs separate at higher temperatures than do AT pairs as there are 3 hydrogen bonds in GC pairs versus only 2 in AT pairs. When the ds DNA has fully separated into 2 ss DNA molecules, ―melting‖ has occurred. When ds DNA melts, its absorbance at 260 nm INCREASES. This phenomenon is known as the HYPERCHROMIC EFFECT. If the temperature is slowly lowered, the complimentary DNA strands realign themselves, re-establish the H-bonds between complimentary bases, and the original ds DNA molecule reforms. Protocol for Determining the Concentration of DNA and RNA. 1. Remove your samples from storage and let them come to room temperature. 2. Set the spectrophotometer to read absorbance at 260 nm and zero the instrument with DEPC-treated sterile water. 3. Place 10 μl of sample in a quartz cuvette and add 990 μl of DEPC-treated sterile water. 4. Record the absorbance. 5. Change the wavelength to 280 nm and record the absorbance. 13 Calculate the concentration of RNA and DNA in your original samples using both methods illustrated in the example. Calculate the purity of your RNA and DNA samples. Is the RNA pure? Is the DNA pure? How can you tell? 14 Molecular Biology Laboratory 4 Restriction Endonuclease Digestion of λ DNA with Hind III. Introduction and Background This experiment will illustrate the method for selectively cutting DNA with a restriction endonuclease, precipitating the cut DNA, separating the DNA fragments by agarose gel electrophoresis, and purifying a selected DNA fragment from the gel using glass milk. The fragment of viral DNA isolated will be cloned into a plasmid vector in subsequent experiments. The source of the viral DNA to be used is from the λ bacteriophage. This virus is harmless to man and therefore makes an excellent source of DNA. The following figure illustrates some of the genes found in the λ genome. Restriction digested DNA from bacteriophage λ is often used as a source of DNA size marker standards on agarose gels to estimate the size of unknown DNA fragments. The figure below is a photograph of an agarose gel on which was separated the DNA fragments resulting from a Hind III digest of lambda DNA. Ethidium bromide was 15 included in the gel. EtBr intercalates between DNA bases and fluoresces when illuminated with long wavelength UV light. Notes on Agarose Gel Electrophoresis of DNA At neutral pH, DNA carries a negative electrical charge due to the acidic phosphoric acid groups of the DNA backbone. If an electrical field is applied to a DNA solution the DNA molecules will migrate towards the positive pole. Agarose is a polysaccharide that when dissolved in water and heated forms a crosslinked array of polysaccharide molecules. DNA migrating through this matrix has its mobility retarded to a degree that is roughly proportional to the logarithm of its size. Therefore, applying an electrical current through an agarose gel containing DNA will separate the DNA molecules according to their size. The molecular weight or size of DNA molecules is customarily reported in kilobase pairs (kbp). The ability of an agarose gel to discriminate between two linear DNA fragments is limited to about 50 kbp. This depends on the concentration of agarose used and the actual size of the fragments. If DNA fragments of known size and similar shape are run on a gel at the same time, they can serve as standard size markers. A standard curve can be plotted of the log molecular weight versus the distance migrated. DNA in solution or in a gel is invisible to the eye. Typically the DNA intercalating molecule ethidium bromide (a potent mutagen) is used to ‗stain‘ the DNA. Ethidium bromide fluoresces under long wavelength UV illumination. EtBr absorbs light in the 320 nm region of the spectrum. When this absorbed energy is lost through fluorescence, photons of less energy and longer wavelength (about 580 nm) are emitted. Light of 580 nm is in the orange-red part of the visible spectrum. 16 The various forms of DNA molecules (linear, covalently closed circular, or nicked circular) can be identified on agarose gels. The following table gives the concentrations of agarose most useful to separate the indicated size ranges of linear DNA to be separated. Agarose % (w/v) Linear DNA Size (kbp) ______________________________________ 0.3 5 – 60 0.6 1 – 20 0.7 0.8 – 10 0.9 0.5 – 7 1.2 0.4 – 6 1.5 0.2 – 3 2.0 0.1 – 2 ______________________________________ Restriction Endonuclease Digestion of λ DNA with Hind III. Obtain a sterile microcentrifuge tube. 1. 2. 3. 4. 5. Pipet 22 μl of sterile, Nano-Pure water into the microfuge tube. Add 3.5 μl (1.0 μg) of λ DNA. Add 3.0 μl of 10X Multicore Restriction Buffer. Add 1.5 μl of Hind III restriction enzyme. Mix by gently pipeting up and down 2 to 3 times. If drops of solution are present on the sides of the tube, centrifuge briefly to pool the solution. Incubate at 37 oC for 30 min. 6. Precipitation of Restricted λ DNA 1. To 25 μl of the restricted DNA, add 2.8 μl of 3M sodium acetate pH 5.2 and 58 μl of 95% ethanol. Mix by gently pipeting several times. Hold at -80 oC for 15 min Centrifuge for 15 min at maximum setting. 2. 3. 4. 17 5. Aspirate the supernatant with a pipet, wash the pellet 1x by resuspending it in 500 μl ethanol, centrifuge for 10 min, then air dry the pellet for 15 min at 37 o C. Add 8 μl of sterile Nano-Pure water to the DNA pellet and allow the DNA to solubilize. Add 2 μl of Gel Loading Solution and mix. 6. 7. Agarose Gel Electrophoresis 1. 2. Load the entire DNA sample into a well of a 0.7% agarose gel . Take note of which lane the sample is loaded into. Run the gel at 100 V for 30 min to 1 h.
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