Molecular Biology Experiment Guide

　　Editor-in-Chief: Yu Bing, Ma Chunquan, Gao Chuanjun, Yang Fengshan

　　Chief Reviewer: Li Haiying

　　College of Life Sciences, Heilongjiang University

　　March 2006

　　Table of Contents

　　Introduction: Molecular Biology Lab Safety Precautions and Required Equipment in Molecular Biology Labs ……………………………………………1

　　Experiment 1: Isolation of Plant Genomic DNA ………………………………6

　　Experiment 2: RNA Isolation ……………………………………………11

　　Experiment 3: Agarose Gel Electrophoresis Detection of DNA/RNA …………………………15

　　Experiment 4: Determination of DNA/RNA Concentration and Purity, and Concentration Adjustment …………21

　　Experiment 5: Polymerase Chain Reaction (PCR) ………………………………24

　　Experiment 6: Random Amplified Polymorphic DNA (RAPD) Reaction …………………28

　　Experiment 7: AFLP Analysis of Beet M14 Strain ………………………………32

　　Experiment 8: Bioinformatics ………………………………………………49

　　Foreword

　　Since its establishment, the College of Life Sciences at Heilongjiang University has successively established majors in Bioengineering, Biotechnology, Biopharmaceutical, and Food Science and Engineering. Molecular biology is a cutting-edge discipline of the 21st century, and experimental operation is an important part of molecular biology. In conjunction with our college's existing equipment and materials, we have set up eight molecular biology experimental projects (including one comprehensive experimental project) for undergraduates, enabling students to master the most basic experimental skills in the field of molecular biology. The writing of this book fully considers the serial operability of experiments, such as from the extraction of genomic DNA and total RNA to agarose gel detection, from DNA concentration adjustment to PCR technology, from molecular marker technology to bioinformatics analysis, etc., thoroughly covering the basic experimental techniques of molecular biology. It is an excellent experimental textbook for undergraduate students of the College of Life Sciences.

　　Introduction: Molecular Biology Lab Safety Precautions and Molecular Biology

　　Required Equipment in the Laboratory

　　I. Safety Precautions

　　Safety is paramount in the laboratory. Molecular biology experiments often involve contact with highly toxic, and even carcinogenic, reagents. Therefore, personnel entering the molecular biology laboratory must understand all safety measures and ensure laboratory operations are conducted under the safest conditions. The following points require special attention:

　　1. Toxic Chemical Reagents

　　Many solvents, such as chloroform, isoamyl alcohol, isobutanol, n-butanol, formaldehyde, and diethyl ether, should be used in a fume hood, with care taken to protect clothing, skin, and respiratory tract. Other reagents may be carcinogens or mutagens, such as ethidium bromide, formamide, diethyl pyrocarbonate (DEPC), etc., and protective measures should be taken during operation, with attention to post-use disposal. Before using any reagent, the instructions for use should be carefully reviewed, especially for reagent kits; only after mastering the relevant knowledge should they be used.

　　2. Radioactivity

　　Some experiments require the use of 32P or other radioactive materials. During experiments, it is crucial to ensure all rules for using such materials are strictly followed; similar care must be taken with radioactive waste.

　　3. Biological Containment

　　Although the development in modern biotechnology has brought tremendous social and economic benefits, the immense power of biotechnology has also brought many unexpected impacts to human society. It can both benefit humanity and potentially be used to create deadly biological weapons, disrupting the peace of the entire human society. On June 30, 1976, the U.S. National Institutes of Health (NIH) formulated and officially published the "Guidelines for Research Involving Recombinant DNA Molecules," which prohibits certain types of recombinant DNA experiments and also stipulated many specific provisions. These provisions mainly include physical containment and biological containment of organisms. Physical containment primarily means: the laboratory must have skilled personnel and correct physical containment equipment, such as negative pressure devices, autoclaves, and biosafety cabinets, etc. Biological containment primarily includes selecting non-pathogenic organisms as cloning vectors for foreign DNA, or performing delicate genetic manipulations on microorganisms to reduce their possibility of survival and reproduction in the environment.

　　4. Equipment-Related Hazards

　　All equipment in the laboratory has potential hazards; incorrect operation can lead to accidents. Therefore, all instruments in the laboratory should be used under the guidance of a teacher, or after reading the instrument's instruction manual.

　　II. Equipment Required in Molecular Biology Laboratories

　　1. Sterilization

　　In molecular biology experiments, many solutions, glassware, and pipette tips require autoclaving, especially in RNA-related experiments, where dry heat and moist heat autoclaving are even more necessary to remove RNase. Electric drying ovens are mainly used for dry heat sterilization; while autoclaves are mainly used for high-pressure moist heat sterilization.

　　2. Centrifuges

　　Almost every molecular biology experiment requires a centrifuge. A molecular biology laboratory needs at least 4 centrifuges: one low-speed (20000 r/min) refrigerated centrifuge, one ultra-speed (20000-80000 r/min) centrifuge, one micro (MINI) centrifuge capable of centrifuging 1.5 mL microcentrifuge tubes, and one large-capacity, low-speed centrifuge for harvesting large volumes of bacterial cultures.

　　3. Refrigeration Equipment

　　At least one regular refrigerator (4℃, -20℃) is needed. If conditions permit, an -80℃ freezer is also required. Many animal, plant, and microbial samples, as well as reagents, need to be stored at low or ultra-low temperatures.

　　An ice machine is also essential, as many reactions are carried out on ice.

　　4. Optical Measurement

　　UV/Visible spectrophotometers are mainly used to detect the concentration and purity of DNA/RNA. Well-equipped laboratories may use DNA/RNA calculators instead of spectrophotometers for more convenience.

　　5. Balances

　　Analytical balances and preparatory balances; the accuracy of the balances should reach one thousandth or one ten thousandth.

　　6. Computers and printers

　　Mainly used for cluster analysis of RAPD and other experiments, and other software analysis.

　　7. Gel electrophoresis apparatus

　　There should be at least one set of horizontal electrophoresis tanks and power supplies to accommodate different molecular weights. Researchers engaged in large-scale sequencing need to be equipped with a sequencing gel apparatus. There should also be a set of vertical electrophoresis units and tanks for polyacrylamide protein gels, and if needed, a special electrophoresis unit for two-dimensional protein gels.

　　8. Incubator (37℃)

　　Used for culturing bacteria

　　9. Temperature-controlled shaker

　　Used for culturing liquid media

　　10. Magnetic stirrer (preferably with heating function)

　　Mainly used for reagent preparation

　　11. Microwave oven

　　Used for melting agar and agarose

　　12. pH meter (acidometer)

　　Reagent preparation

　　13. Micropipettes ranging from 0.1 μL to 5000 μL

　　Generally, they are adjustable pipettes, with adjustable ranges of 0.1 μL—2.5 μL, 5 μL—10 μL, 10 μL—100 μL, 20 μL—200 μL, 100 μL—1000 μL, and 1000 μL—5000 μL respectively.

　　14. PCR machine (thermal cycler)

　　Used for PCR amplification reaction

　　15. Tissue illumination incubator

　　Used for tissue culture

　　16. UV transilluminator

　　UV observation of gel plates

　　17. Vortex mixer (Vortex)

　　Mixing trace reagents

　　18. Water bath apparatus

　　At least two water baths are required; DNA extraction and many reactions need to be carried out in a water bath.

　　19. Pure water system

　　All water used in molecular biology must be pure water, with resistivity reaching MΩ level.

　　20. Glassware and plasticware

　　Beakers, flasks, reagent bottles, measuring cylinders, pipettes, stirring rods, grinders, centrifuge tubes (1.5mL, 0.5mL), PCR tubes, pipette tips (Tip), etc.

　　21. Corresponding molecular biology reagents and kits

　　III. How to Use Some Commonly Used Precision Instruments

　　1. How to use micropipettes

　　Demonstrated using an Eppendorf micropipette as an example

　　2. How to use refrigerated centrifuges

　　Demonstrated using a Beckman Microfuge R refrigerated centrifuge as an example

　　3. How to use PCR machines

　　Demonstrated using a PE9700 PCR machine as an example

　　4. How to use pure water systems

　　Demonstrated using a MiLLi-Q pure water system as an example

　　IV. Thinking Questions

　　1. What safety precautions should be observed in molecular biology experiments?

　　2. Correct use of micropipettes.

　　Experiment 1: Plant Genomic DNA Isolation

　　I. Related Knowledge

　　Isolating plant DNA is a fundamental requirement in molecular biology experiments. Different research objectives have varying demands for DNA purity and quantity. For example, when constructing genomic DNA libraries for screening plant genes or other genetic markers like RFLP, high molecular weight, high-purity DNA is required; whereas for genetic analysis, the purity requirements for DNA can be lower.

　　Generally, a good DNA isolation procedure should meet the following three main criteria: (1) The purity of the obtained DNA should meet the requirements for downstream operations. For RFLP analysis, DNA purity requires complete enzymatic digestion by restriction endonucleases and successful transfer to a membrane for Southern hybridization; DNA for PCR analysis should not contain contaminants that interfere with the PCR reaction; (2) The obtained DNA should be intact, yielding highly accurate and reproducible migration patterns upon electrophoresis; (3) The obtained DNA should be in sufficient quantity.

　　If large-scale screening of plants is performed, the operating procedure should also be fast, simple, inexpensive, and avoid the use of toxic reagents as much as possible.

　　II. Experimental Principle

　　Plant DNA extraction procedures should include the following: First, the cell wall must be crushed (or digested) to release the intracellular contents. Common cell disruption methods for isolating total genomic DNA include swelling plant tissue and then grinding it into a fine powder; or rapidly freezing fresh plant tissue in dry ice or liquid nitrogen, then grinding it into a powder with a mortar.

　　Second, the cell membrane must be disrupted to release DNA into the extraction buffer. This step is usually accomplished by detergents such as SDS or CTAB. Detergents also protect DNA from degradation by endogenous nucleases. The extraction buffer typically also contains EDTA, which chelates magnesium ions—a cofactor required by most nucleases.

　　Finally, once DNA is released, the extent of shear damage must be minimized. Vigorous shaking or rapid aspiration through small pipette tips can break high molecular weight DNA in solution.

　　Separating high molecular weight DNA is only part of the job. Because crude extracts often contain a large amount of impurities such as RNA, proteins, and polysaccharides, these impurities are sometimes difficult to remove from DNA. Most proteins can be denatured and precipitated by chloroform or phenol treatment, and most RNA can be removed by treated RNase. However, polysaccharide impurities are generally difficult to remove; when these impurities are at high concentrations, some DNA purification kits are often used to further purify DNA.

　　III. Experimental Methods

　　1. Add 5 mL of 2×CTAB (Cetyltrimethylammonium bromide) to a 10 mL centrifuge tube, preheat at 60℃, and after preheating, add 10 μL of 0.2% β-mercaptoethanol. β-mercaptoethanol acts as an antioxidant.

　　2. Preheat the pestle and mortar with liquid nitrogen, put 0.5-1.5 g of leaves into the mortar, grind into powder, transfer to the centrifuge tube in ①, gently rotate the centrifuge tube to uniformly separate the plant tissue in the extraction buffer, and incubate in a 60℃ water bath shaker for 30 min.

　　3. Add an equal volume of 5 mL chloroform, shake in an air shaker for 15 min to make the mixture in the tube an emulsion. At this time, proteins fully contact chloroform and separate from nucleic acids. Centrifuge at 4000 rpm for 15 min at room temperature for phase separation. The organic phase containing protein cell fragments is at the bottom of the tube, and the upper layer is the aqueous phase containing nucleic acids.

　　4. After centrifugation, use a truncated tip (to avoid damaging long DNA strands) to transfer the supernatant to a new centrifuge tube. Slowly mix with 2/3 volume, i.e., 3.3 mL, of pre-cooled isopropanol. Centrifuge at 8000 rpm for 10 min at 20℃ to precipitate DNA.

　　5. Remove the supernatant, dry (ventilate and dry using a sterile workbench), and add 1 mL TE to dissolve the precipitate.

　　6. Add 5 μL RNase (10mg/mL) and incubate at 37℃ for 45 min to remove RNA from DNA.

　　7. Add an equal volume (1 mL) of phenol-chloroform-isoamyl alcohol (25:24:1), slowly mix in an air shaker at room temperature for 10 min, then centrifuge at 4000 rpm for 10 min at room temperature to further remove proteins. Use a truncated 200 μL tip to transfer the supernatant to a new centrifuge tube.

　　8. Add 1/2 volume (0.5 mL) of phenol-chloroform-isoamyl alcohol (25:24:1), slowly mix in a shaker for 10 min, centrifuge at 4000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube.

　　9. Add an equal volume (0.8-1 mL) of chloroform, shake for 10 min, centrifuge at 4000 rpm for 10 min, and transfer the supernatant to a new tube. This step removes residual phenol from the aqueous phase.

　　10. Add 1/10 volume (0.1 mL) of 3M NaAc, 2.5 times volume (2.5 mL) of cold ethanol, slowly mix. DNA precipitate will separate out. Centrifuge at 4000 rpm for 10 min.

　　11. Discard the supernatant, dry for 30-60 min, add 0.5-1 mL TE, dissolve and store at 4℃.

　　IV. Key Experimental Points

　　1. CTAB solution will precipitate below 15℃, so it must be preheated before adding it to frozen plant material.

　　2. Under optimal conditions, DNA-CTAB precipitate is white and fibrous, and can be easily hooked out of the solution in one go. However, DNA precipitate from certain plant species may contain impurities, especially polysaccharides, making the DNA precipitate flocculent or gelatinous. In this case, a brief centrifugation may be required to obtain the DNA-CTAB precipitate.

　　3. Although saturated phenol can effectively denature proteins, phenol cannot completely inhibit RNase activity, and phenol can dissolve poly(A)-containing mRNA. Using a mixture of phenol and chloroform can alleviate these two phenomena, and an appropriate amount of isoamyl alcohol can be added (phenol-chloroform-isoamyl alcohol = 25:24:1). Isoamyl alcohol acts as an antifoaming agent and compacts the protein layer, resulting in better separation of the aqueous and organic phases.

　　V. Required Instruments and Consumables

　　1. 1.5mL Centrifuge Tube

　　2. 60℃ Water Bath, 37℃ Water Bath

　　3. Mortar and Pestle

　　4. Air Shaker

　　5. Refrigerated Centrifuge

　　6. Clean Bench

　　7. Refrigerator

　　8. Truncated 10 μL Tip

　　9. Truncated 200 μL Tip

　　10. Absorbent Paper

　　11. Liquid Nitrogen Tank

　　VI. Required Reagents

　　1. CTAB (Cetyltrimethylammonium bromide)

　　2. β-mercaptoethanol

　　3. Liquid Nitrogen

　　4. Chloroform

　　5. Cold Isopropanol

　　6. TE Buffer

　　7. RNase

　　8. Phenol-Chloroform-Isoamyl Alcohol (25:24:1)

　　9. 3M NaAc

　　10. Cold Ethanol

　　VII. Reagent Preparation

　　1. 1M Tris-HCl, pH=8.0

　　Tris-HCl: 121.1g 60.55g

　　Conc. HCl: 42mL 21mL

　　Add H2O to: 1L 500mL

　　→Autoclave for 15min

　　2. 0.5M EDTA Na2·2H2O, pH=8.0

　　EDTA Na2·2H2O: 186.1g 93.05g 55.83g

　　Add H2O to: 1L 500mL 300mL

　　Adjust pH to 8.0 with NaOH (approx. 20g NaOH pellets needed)

　　→Autoclave

　　3. 2×CTAB

　　40.9g NaCl; 10g CTAB, 50mL 1M Tris-HCl (pH=8.0)

　　Make up to 500mL with 20mL 0.5M EDTA (pH 8.0), then autoclave.

　　4. TE Buffer

　　1M Tris-HCl 5mL + 0.5M EDTA 1mL → add water to make up to 500mL, then autoclave.

　　5. 3M NaAC (pH=5.2)

　　Dissolve 102g sodium acetate trihydrate in 200mL water, adjust pH to 5.2 with glacial acetic acid, add water to make up to 250mL, then autoclave.

　　6. 0.2% Mercaptoethanol

　　Add 0.2mL mercaptoethanol to 100mL water.

　　7. 20×SSC

　　NaCl: 175.3g 87.65g

　　Sodium Citrate: 88.2g 44.1g

　　10N NaOH: Adjust pH to 7.0 with a few drops

　　ddH2O: 1L 500mL

　　8. 10mg/mL RNase A

　　Dissolve 10mg RNaseA in 1mL 2×SSC (0.1mL 20×SSC made up to 1mL), boil in water for 10 minutes, cool, aliquot and store at -20℃.

　　Eight, Discussion Questions

　　1. What is the role of CTAB or EDTA in the extraction buffer?

　　2. How to remove proteins and RNA from DNA impurities?

　　3. What are the roles of phenol, chloroform, and isoamyl alcohol in the DNA purification steps? Why are they used alternately?

　　Experiment Two: RNA Isolation

　　One, RNA and RNA-free Environment

　　Different types of RNA exist in plant cells. rRNA accounts for 70% of total RNA; tRNA is also relatively abundant in cells (15%), and mRNA content is 1-5% of total cellular RNA. Most eukaryotic mRNA has a poly-A tail at its 3' end.

　　When performing RNA operations, special attention must be paid to RNA degradation by RNases. Since RNases are present in all living organisms and are highly tolerant enzymes, traditional high-temperature sterilization methods cannot inactivate them. RNase contamination can come from internal sources, such as certain plant tissues like roots where RNase content is particularly high, or from external sources, such as glassware, buffers, and the operator's skin. Human skin surface contains a large amount of RNase. Therefore, RNA operations should be carried out in an RNase-free environment as much as possible. Specific measures are as follows:

　　1. Before RNA isolation, place all glassware, sampling scissors, forceps, etc., in an oven and bake at 300℃ for 4h or 180℃ for 8h for sterilization.

　　2. Use diethyl pyrocarbonate-treated water (DEPC-SDW) for solution preparation and autoclave for 20min. DEPC is a strong RNase inhibitor. DEPC-SDW preparation is as follows:

　　Add 0.2mL DEPC stock solution to 100mL water, mix thoroughly on a shaker and allow to act for several hours at room temperature, then autoclave for 15min.

　　3. All plasticware such as tips, centrifuge tubes, PCR tubes, etc., should be treated with 0.05% DEPC-H2O: fill with DEPC-H2O, incubate at 37℃ for 2 hours, rinse with DEPC-SDW, then dry bake at 100℃ for 15 minutes and autoclave for 15 minutes.

　　4. Human sweat contains RNases, so gloves should be worn and changed frequently during all steps. Commonly used laboratory equipment such as pipettes should be soaked in alcohol and air-dried before use.

　　II. Experimental Principle

　　Total RNA extraction is performed using a one-step method (TRIzol reagent). The reagent contains phenol, guanidine, and thiocyanate, and is an improved RNA extraction method developed by Chomczyanski and Sacchi. During sample homogenization and dissolution, TRIzol reagent can break cells, disrupt nucleoprotein complexes, allow RNA to be released smoothly into the buffer, and simultaneously maintain RNA integrity. Since RNA is unstable under alkaline conditions, the system remains acidic to neutral throughout the RNA extraction process. Under acidic conditions, DNA rarely dissociates; DNA, along with proteins, denatures and is pelleted by centrifugation. At this point, the solution separates into an aqueous phase and an organic phase, and RNA can be completely preserved in the aqueous phase. After extracting the aqueous phase, isopropanol is added to precipitate RNA, and finally, 75% ethanol is used to wash the RNA.

　　III. Experimental Methods

　　(I) Homogenization and Phase Separation

　　1. Place 1mL TRIzol RNA extraction solution in a glass homogenizer.

　　2. Grind on ice.

　　3. Transfer the homogenized solution to a 1.5mL centrifuge tube and let stand at room temperature for 5 minutes.

　　4. Add 0.2mL chloroform, close the lid, and shake vigorously by hand (or vortex) for 15 seconds.

　　5. Let stand at room temperature for another 2-3 minutes.

　　Centrifuge at 12000g for 15min at 6.4℃. The centrifuge tube is divided into three phases: the upper layer is the RNA liquid phase, the middle layer is the DNA and fragmented tissue phase, and the lower layer is the phenol-chloroform phase.

　　7. Transfer the supernatant RNA liquid phase into a new centrifuge tube.

　　(II) RNA Precipitation

　　8. After mixing with 0.5mL isopropanol, let it stand at room temperature for 10 minutes (wash chloroform, precipitate RNA).

　　9. Centrifuge at 12000g for 10 minutes at 4℃.

　　(III) Washing

　　10. Discard the supernatant, then add 1mL of 75% ethanol and vortex to wash RNA (to remove isopropanol).

　　11. Centrifuge at 7500g for 5 minutes at 4℃.

　　(IV) Re-dissolving RNA

　　12. Remove the ethanol.

　　13. Dry for 10 minutes.

　　14. Dissolve the RNA pellet with 50 μL of DEPC-SDW, and incubate at 55-60℃ for 10min.

　　15. Add 3 times the volume of absolute ethanol (150 μL) and store at -20℃ or -80℃.

　　IV. Required Instruments and Consumables

　　1. 180℃ Oven

　　2. 4℃ Centrifuge

　　3. Glass Mortar and Pestle

　　4. Centrifuge Tube

　　5. Pipette

　　6. Pipette Tips

　　7. Lunch Box

　　8. Filter Paper

　　9. Glassware

　　10. Autoclave

　　11. Gauze

　　12. Disposable Gloves

　　13. Water Bath

　　14. Vortex

　　15. White Ceramic Plate

　　V. Required Reagents

　　1. TRIzol Reagent

　　2. Chloroform

　　3. Isopropanol

　　4. 75% Ethanol (prepared with DEPC-SDW)

　　5. DEPC-H2O

　　VI. Discussion Questions

　　1. What is the role of TrizoL reagent in the experiment?

　　2. During the experiment, after adding chloroform, into which three phases is the solution divided?

　　3. How to achieve an as much as possible RNase-free environment?

　　Experiment 3: Agarose Gel Detection of DNA/RNA

　　I. Experimental Principle

　　After isolating total RNA or partial RNA from cells, electrophoresis can be used to resolve RNA molecules based on their respective migration, which is a critical step in detecting whether RNA molecules are degraded.

　　Electrophoresis is currently the most commonly used technique for separating and purifying DNA/RNA fragments. When a prepared "gel," a porous support medium containing electrolytes, is placed in an electrostatic field, DNA/RNA molecules will migrate towards the anode because DNA/RNA molecules carry negatively charged phosphate residues along both sides of their double helix backbone. As the length of DNA/RNA increases, the ratio between the driving force from the electric field and the resistance from the gel decreases, resulting in different migration rates for DNA/RNA fragments of different lengths. Therefore, DNA/RNA molecules can be separated based on their size.

　　Based on the material used to prepare the gel, gel electrophoresis can be divided into two subclasses: agarose gel electrophoresis and polyacrylamide gel electrophoresis. Polyacrylamide separates small DNA/RNA fragments (5-500bp) with extremely high resolving power, allowing DNA fragments differing by just 1bp to be separated. Its disadvantage is that preparation and operation are relatively difficult. Agarose gel has lower resolving power than polyacrylamide gel but a wider separation range. Agarose gels of various concentrations can separate DNA/RNA fragments ranging from 200bp to nearly 50kb in length.

　　(I) Agarose Gel

　　1. Agarose Concentration

　　Gels with different concentrations can resolve a wide range of DNA molecules.

　　2. Presence of EB Dye

　　Ethidium bromide (EB) is an acridine dye that emits fluorescence under UV light. When DNA samples are electrophoresed in an agarose gel, the EB in the agarose gel intercalates into the DNA molecules to form a fluorescent complex, enhancing the fluorescence emitted by DNA tens of times. After electrophoresis, the DNA in the agarose can be directly detected under UV light.

　　Separation Range of Gels Containing Different Amounts of Agarose

　　Agarose Content in Gel [%(W/V)] Separation Range of DNA/RNA Molecules (kb)

　　0.35—60

　　0.61—20

　　0.70.8—10

　　0.90.5—7

　　1.20.4—6

　　1.50.2—3

　　2.00.1—2

　　3. Composition of Electrophoresis Buffer

　　There are several different buffers available for electrophoresis, namely Tris-acetate (TAE), Tris-borate (TBE), or Tris-phosphate (TPE), with a concentration of about 50 mmol/L and a pH of 7.5-7.8. All these buffers contain EDTA. These buffers are usually prepared as concentrates and stored at room temperature.

　　Preparation of commonly used electrophoresis buffers

　　Buffer working solution concentrated stock solution (per liter)

　　Tris-acetate (TAE) 1×: 0.04 mol/L Tris-acetate

　　0.001 mol/L EDTA 50×: 242g Tris base

　　57.7 mL Glacial acetic acid

　　100 mL 0.5 mol/L EDTA (pH 8.0)

　　Tris-phosphate (TPE) 1×: 0.09 mol/L Tris-phosphate

　　0.002 mol/L EDTA 10×: 108g Tris base

　　15.5 mL 85% Phosphoric acid

　　40 mL 0.5 mol/L EDTA (pH 8.0)

　　Tris-borate (TBE) 0.5× 0.045 mol/L Tris-borate

　　0.001 mol/L EDTA 5×: 54g Tris base

　　27.5g Boric acid

　　20 mL 0.5 mol/L EDTA (pH 8.0)

　　4. Loading buffer:

　　Buffer type 6× Buffer storage temperature

　　I 0.25% Bromophenol blue

　　0.25% Xylene cyanol FF

　　40% (W/V) Sucrose aqueous solution 4℃

　　II 0.25% Bromophenol blue

　　0.25% Xylene cyanol FF

　　15% Ficoll aqueous solution Room temperature

　　III 0.25% Bromophenol blue

　　0.25% Xylene cyanol FF

　　30% Glycerol aqueous solution 4℃

　　IV 0.25% Bromophenol blue

　　40% (W/V sucrose aqueous solution) 4℃

　　V

　　(Alkaline loading buffer) 300 mmol/L NaOH

　　6 mmol/L EDTA

　　18% Ficoll aqueous solution

　　0.15% Bromocresol green

　　0.25% Xylene cyanol FF

　　4℃

　　Loading buffer can increase sample density to ensure DNA enters the sample wells uniformly, and it can also color the sample, making the loading operation more convenient. The migration rate of bromophenol blue in agarose gel is approximately 2.2 times that of xylene cyanol FF. The migration rate of bromophenol blue in agarose gel is approximately the same as that of 300 bp double-stranded linear DNA, while the migration rate of xylene cyanol FF in agarose gel is the same as that of 4 kb double-stranded linear DNA. The choice of which loading buffer to use is purely a matter of personal preference.

　　5. DNA fragment size marker

　　DNA fragment size markers are commonly called DNA Markers. This experiment uses Gene RulerTM DNA Ladder Mix as the DNA fragment size marker, consisting of 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1031 bp, 1200 bp, 1500 bp, 2000 bp, 2500 bp, 3000 bp, 3500 bp, 4000 bp, 5000 bp, 6000 bp, 8000 bp, and 10000 bp. DNA markers can not only serve as a marker for DNA fragment length in gels but also as a control for electrophoresis.

　　II. Experimental Methods

　　1. Dilution of 50× TAE: To prepare 50 mL of 1× TAE, take 1 mL of 50× TAE and add 49 mL of water to a final volume of 50 mL.

　　2. Preparation of 1% agarose gel solution: Dissolve 0.2g of agarose in 20mL of TAE, heat for a short time until all agarose melts, cool the solution to 60℃, add 2 μL of 10 mg/mL EB solution, making the final concentration of EB 1 μg/mL.

　　3. The electrophoresis tank used for RNA electrophoresis should be washed clean with detergent, then rinsed with water, dried with ethanol, filled with 3% H2O2 solution, left at room temperature for 10 minutes, then rinsed with DEPC-SDW. The comb should be treated similarly.

　　4. Seal the gel tray with tape and place the comb.

　　5. Pour the warm agarose into the gel tray. The gel thickness should be between 3-5mm. Allow to solidify for 20-60 min.

　　6. After the gel has completely solidified, carefully remove the comb and tape, place the gel tray inside the electrophoresis tank, with the sample well side facing the cathode (black pole).

　　7. Pour an appropriate amount of TAE buffer into the electrophoresis tank. The buffer should typically be 1 cm above the gel surface.

　　8. Mix the RNA sample with the loading buffer (10 μL RNA sample + 2 μL 6× loading buffer), and load 12 μL of the sample into the sample wells using a pipette.

　　9. Properly connect the electrophoresis tank to the power supply, set the constant voltage to 75V, and the current typically to 50mA.

　　10. After electrophoresis, observe on a UV transilluminator. Three bands of intact extracted RNA can be seen: 5kb 28S rRNA, 2kb 18S rRNA, and 0.1-0.3kb 5S rRNA and tRNA. It can be observed that the content of 28S rRNA is approximately twice that of 18S rRNA, indicating good integrity of the total RNA with no degradation.

　　III. Precautions

　　Purification Treatment of Ethidium Bromide Solution:

　　Ethidium bromide is a strong mutagen, has moderate toxicity, and can cause cancer. Always wear gloves when using this dye.

　　1. Purification treatment of concentrated ethidium bromide solution (concentration > 0.5mg/mL).

　　① Add sufficient water to reduce the EB concentration to below 0.5mg/mL.

　　② Add 1 volume of 0.5moL/L KMnO4, mix carefully, then add 1 volume of 2.5moL/L HCl, mix carefully, and let stand at room temperature for several hours.

　　③ Add 1 volume of 2.5moL/L NaOH, mix carefully, then the solution can be discarded.

　　2. Purification treatment of dilute ethidium bromide solution (e.g., electrophoresis buffer containing 0.5μg-1μg/mL ethidium bromide).

　　① For every 100mL of solution, add 100mg powdered activated carbon.

　　② Let stand at room temperature for 1 hour, shaking occasionally.

　　③ Filter the solution with filter paper, discard the filtrate.

　　④ Seal the filter paper and activated carbon in a plastic bag and dispose of as hazardous waste.

　　IV. Required Instruments and Consumables

　　1. Electrophoresis apparatus 6. Micropipette

　　2. Electrophoresis tank 7. UV transilluminator or UV lamp

　　3. Balance 8. Various glassware

　　4. Magnetic stirrer (beakers, volumetric flasks, and wide-mouth bottles of various specifications)

　　5. pH meter 9. UV protective glasses

　　V. Required Reagents

　　1. EDTA—Na2·2H2O (Ethylenediaminetetraacetic acid disodium salt)

　　2. Ethidium bromide (EB)

　　3. Agarose

　　4. Tris (Tris(hydroxymethyl)aminomethane)

　　5. Glacial acetic acid

　　6. Bromophenol blue

　　7. Sucrose or glycerol

　　8. Xylene cyanol FF

　　VI. Reagent Preparation

　　1. Loading buffer: (6×) Type IV

　　0.25% bromophenol blue, 40% (W/V) sucrose aqueous solution

　　To prepare 50mL, add 0.125g bromophenol blue, 20g sucrose, mix well, make up to 50mL, and store at 4°C.

　　2. TAE, 50× concentrated stock solution:

　　Tris 242g, glacial acetic acid 57.1mL, 100mL 0.5moL/L EDTA, pH=8.0, add 600mL water, stir vigorously, make up to 1000mL.

　　3. 0.5moL/L EDTA, pH=8.0

　　Add 186.1g EDTA—Na2·2H2O to 800mL water, stir vigorously on a magnetic stirrer, adjust the solution's pH to 8.0 with NaOH (EDTA—Na2·2H2O will only completely dissolve when the solution's pH is close to 8.0), then make up to 1 L, aliquot and autoclave for future use.

　　4. Ethidium bromide stock solution (10mg/mL):

　　Add 1g ethidium bromide to 100mL water, stir for several hours with a magnetic stirrer, then transfer to a brown bottle, and store at 4°C.

　　VII. Discussion Questions

　　1. What is the function of the loading buffer?

　　2. What is the function of adding EB to agarose?

　　3. During electrophoresis, why do DNA/RNA molecules move towards the anode?

　　Experiment 4 Determination of DNA/RNA Concentration and Purity, and Concentration Adjustment

　　I. Experimental Principle

　　In molecular biology experiments, after extracting DNA or RNA, it is often necessary to determine their purity and concentration. Only after the purity meets the standard and the concentration is adjusted to the desired level can the next experiment be carried out.

　　Well-equipped laboratories use DNA/RNA calculators, which can conveniently and quickly determine DNA/RNA concentration and purity, but DNA/RNA calculators are often expensive. General laboratories often use a UV spectrophotometer to determine DNA/RNA concentration.

　　1. For DNA, its purity and concentration can be determined based on its ultraviolet absorption values A260, A280, A310 at 260, 280, 310nm.

　　For dsDNA: 1.0A260=50μg/mL

　　ssDNA: 1.0A260=33μg/mL

　　The A260/280 ratio of pure DNA solution should be 1.8±0.1; if higher than 1.8, there may be RNA contamination; if lower than 1.8, there is protein contamination.

　　The A310 value is the background; if the salt concentration is high, the A310 value is also high.

　　2. For RNA, its purity and concentration can be determined based on its ultraviolet absorption values A230, A260, A280 at 230, 260, 280nm.

　　1.0A260=40μg/mL

　　A260/A280 should be between 1.7~2.0

　　If the A260/280 ratio is too low, it indicates that the RNA sample may be contaminated with protein or phenol. The A260/A230 ratio should be greater than 2.0, otherwise, it may be contaminated with guanidine thiocyanate (a component of TRizol reagent).

　　II. Experimental Methods

　　1. Before measurement, the quartz cuvette must be immersed in a 1:1 solution of concentrated hydrochloric acid:methanol for 1 hour, then thoroughly rinsed with DEPC-SDW.

　　2. Take the RNA sample finally extracted in Experiment 3 as an example.

　　The original RNA sample contained 200μL of water. Add 2μL of 3moL/L pH=5.2 sodium acetate solution to the sample to make its final concentration 0.03moL/L. Mix thoroughly for precipitation, centrifuge at 12000g for 5min at 4℃, discard the supernatant, wash the precipitate with pre-cooled 70% ethanol, air dry, then dissolve the RNA with 20μL DEPC-SDW.

　　3. Dilute the sample to be tested 1:1000 with DEPC-SDW (add 2μL sample to 1.998 mL DEPC-SDW).

　　4. Measure the A260, A280, and A230 of the sample separately.

　　5. Calculate the A260/A280 and A260/A230 ratios of the sample separately.

　　III. Required Equipment

　　1. Centrifuge

　　2. Micropipette

　　3. UV Spectrophotometer

　　IV. Required Reagents

　　1. Concentrated Hydrochloric Acid

　　2. Methanol

　　3. Sodium Acetate

　　4. DEPC-SDW

　　V. Reagent Preparation

　　Preparation of 3moL/L Sodium Acetate (pH5.2) 250mL Solution:

　　Dissolve 102.025g of sodium acetate trihydrate in 200mL of DEPC-SDW, adjust the pH to 5.2 with glacial acetic acid, add 25μL of original DEPC solution, then make up to 250mL, let stand overnight, and autoclave for 15min.

　　VI. Discussion Questions

　　1. How to determine the concentration of DNA or RNA solution?

　　2. Based on the measured concentration of DNA or RNA, calculate how much μL of water should be added to the solution if the solution concentration is adjusted to 0.1μg/μL?