Experiment 5: Preparation and Transformation of E. coli Competent Cells

I. Experimental Principle

In vitro ligated recombinant DNA molecules can only replicate, proliferate, and express in large quantities after being introduced into suitable host cells. Research has found that treating E. coli cells with Ca2+-containing solutions makes them more susceptible to absorbing foreign DNA. The process of E. coli absorbing foreign DNA is called transformation. The state where bacteria are susceptible to absorbing foreign DNA is called competency. Physical and chemical methods can induce cells into a competent state, such as electroporation and CaCl2 method.

The CaCl2 method is currently a commonly used laboratory method for preparing competent cells. Its principle is to place bacteria in a 0°C, hypotonic CaCl2 solution, causing the cells to swell into spherical shapes. The permeability of the cell membrane changes, and foreign DNA attaches to the cell membrane surface. A brief heat shock treatment at 42°C promotes the cell's absorption of the DNA complex. Host cells are generally mutant strains with a defective restriction-modification system, meaning they lack restriction endonucleases and methylases. Plasmid vectors carry a specific antibiotic resistance gene, such as the ampicillin resistance gene (AP+). If transformed cells are plated on an ampicillin-containing plate, only those cells containing the transformed plasmid can survive and grow. This allows for the selection of clones containing the plasmid.

In this experiment, E.coli JM109 strain is used as the recipient cell. The recipient bacteria are treated with CaCl2 to make them competent, and then co-incubated with pUC18 plasmid for transformation. All transformed recipient cells are appropriately diluted and cultured on an ampicillin-containing plate medium. Only transformants can survive, while untransformed recipient cells die due to their inability to resist ampicillin. After amplification, the transferred plasmid DNA can be isolated and extracted for experiments such as electrophoresis analysis, restriction endonuclease mapping, and DNA sequencing.

II. Instruments and Reagents

1. Instruments:

Constant temperature shaker, electric constant temperature incubator, electric constant temperature water bath, clean bench, spectrophotometer, high-speed refrigerated centrifuge, desktop centrifuge.

2. Materials

E.coli JM109 recipient bacteria, plasmid pUC18.

3. Reagents:

LB broth (1L):

Tryptone 10g

Yeast extract 5g

NaCl 10g (high salt) or 5g (low salt)

Ampicillin 100mg/ml

Dissolve 10g tryptone, 5g yeast extract, and 5g NaCl in 1L of double-distilled water in an Erlenmeyer flask and autoclave. After cooling to 55℃, add 1.5ml of 100mg/ml ampicillin to the solution. Then store at 4℃ for later use.

LB agar medium:

Tryptone 10g

Yeast extract 5g

NaCl 10g or 5g,

Agar 13g

Add the above ingredients sequentially to 1L of double-distilled water in an Erlenmeyer flask and autoclave. Once the medium cools to 50℃, remove it and gently swirl to ensure the dissolved agar is uniformly distributed throughout the medium solution. Add ampicillin as needed, then the medium can be directly poured from the Erlenmeyer flask to prepare plates. A 90mm diameter petri dish requires approximately 30-50ml of medium. After the medium has completely solidified, invert the plates and store at 4℃ for later use.

Other reagents: 0.1M CaCl2 solution, sterile distilled water

III. Operating Procedures

1. Preparation of Competent Bacteria:

(1) Pick a single colony from an E.coli JM109 bacterial plate with an inoculation loop, inoculate it into a test tube containing 5ml LB liquid medium, and incubate overnight (12-16h) at 37℃ with shaking at 220 rpm.

(2) Inoculate 0.5 ml of overnight culture into an Erlenmeyer flask containing 50ml LB liquid medium, and incubate at 37℃ with shaking at 220 rpm for 2-3h. Measure the OD600 value every 20-30min until it reaches 0.3-0.4. Under sterile conditions, transfer the bacterial culture to 1.5ml centrifuge tubes, 1ml bacterial culture per tube, determining the number of tubes as needed.

(3) Centrifuge at 4℃, 12000 rpm for 2 min to pellet the bacteria.

(4) Discard the supernatant, invert the centrifuge tube on filter paper for 1 min to drain any residual trace medium. Add 1ml of ice-cold 0.1mol/L CaCl2 to each tube to resuspend the cells, and incubate on ice for 30 min.

(5) 4℃, 12000 rpm, 2 min.

(6) Discard the supernatant, invert the centrifuge tube for 1 min to drain any residual liquid. Add 100 ul of ice-cold 0.1 mol/L CaCl2 to each tube to re-suspend the cells (handle gently during re-suspension), forming the competent cell suspension.

2. Bacterial Transformation:

(1) Take 3 sterile Eppendorf tubes, label them 1, 2, and 3 respectively, and add the following components: Add 10-20ng of plasmid DNA (volume less than 10ul), and include two control groups:

No. 1: Recipient cell control group: 100ul competent cell suspension + 2ul sterile ddH2O

No. 2: Plasmid DNA control group: 100ul 0.1mol/L CaCl2 + 2ul pUC19

No. 3: Normal transformation group: 100ul competent cell suspension + 2ul pUC19

(2) Gently swirl the centrifuge tubes to mix the contents, then incubate on ice for 30min to promote the adsorption of DNA molecules onto the surface of competent cells.

(3) Transfer to a 42℃ water bath for 2min to enhance the effect of CaCl2 through heat stimulation, causing channels to form in the cell membrane, facilitating the adsorption and entry of DNA molecules, and improving transformation efficiency.

(4) Quickly transfer to ice for 2min to cool and repair the cell membrane.

(5) Add 600u of antibiotic-free LB liquid medium, shake well, and incubate at 37℃, 220 rpm for 60min. This allows the recipient bacteria to recover normal growth and express the Ampicillin resistance gene.

(6) Spread 200ul of bacterial solution onto an Ampicillin-containing LB plate, place the plate on a sterile bench until the liquid is absorbed, and then invert and culture in a 37℃ incubator for 12-16h before observing the results. Store the rest at 4℃. If no colonies grow on the plate, it can be left in a 37℃ incubator for further cultivation. At the same time, centrifuge the remaining 500ul of bacterial solution, discard most of the supernatant, leaving about 100ul, resuspend by pipetting, and then spread all of it onto an Ampicillin-containing LB plate and incubate at 37℃.

(7) Observe and record the number of transformants, and calculate the transformation efficiency.

IV. Precautions:

1. All vessels used in the experiment must be sterilized to prevent contamination by miscellaneous bacteria and exogenous DNA.

2. Pay attention to sterile operation during the experiment; solution transfer, aliquoting, etc., should all be performed on a sterile ultra-clean workbench.

3. A control group must be set up to check for contamination during the experiment.

4. The entire experimental process should be kept on ice.

5. Select cells in the logarithmic growth phase; OD600 should not be higher than 0.6.

Experiment 6: Genomic DNA Extraction from Animal Tissue Cells

I. Experimental Principle

All nucleated cells of eukaryotes (including cultured cells) can be used to prepare genomic DNA. Eukaryotic DNA exists in the nucleus in the form of chromosomes. Therefore, the principle of DNA preparation is to separate DNA from proteins, lipids, and carbohydrates, while maintaining the integrity of the DNA molecule. The general process for DNA extraction involves digesting and decomposing proteins in dispersed tissue cells with a solution containing SDS (sodium dodecyl sulfate) and proteinase K, then extracting proteins with phenol and chloroform/isoamyl alcohol, and finally precipitating the obtained DNA solution with ethanol to separate DNA from the solution.

An important characteristic of Proteinase K is its ability to maintain high activity in the presence of SDS and EDTA (disodium ethylenediaminetetraacetate). In the reaction system for DNA extraction after homogenization, SDS can disrupt cell membranes and nuclear membranes, separating tissue proteins from DNA, while EDTA inhibits DNase activity in cells; Proteinase K can degrade proteins into small peptides or amino acids, allowing the complete separation of DNA molecules.

II. Instruments and Reagents

1. Instruments:

Constant temperature water bath, benchtop centrifuge, UV spectrophotometer (GeneQuant), pipettor, glass homogenizer, centrifuge tubes (sterile), tips (sterile)

2. Reagents:

(1) Cell Lysis Buffer:

Tris (pH8.0) 100 mmol/L

EDTA (pH 8.0) 500 mmol/L

NaCl 20 mmol/L

SDS 10%

Pancreatic RNase 20ug/ml

(2) Proteinase K: Weigh 20mg Proteinase K and dissolve in 1ml sterile double-distilled water, store at -20℃ for later use.

(3) TE Buffer (pH 8.0): Autoclaved, store at room temperature.

(4) Phenol:Chloroform:Isoamyl Alcohol (25:24:1),

(5) Isopropanol, cold absolute ethanol, 70% ethanol, sterile water.

III. Operating Procedures

1. Take 0.1g (0.5cm³) of fresh or frozen animal tissue block, cut into pieces as small as possible. Place in a glass homogenizer, add 1ml of cell lysis buffer, and homogenize until no tissue blocks are visible. Transfer to a 1.5ml centrifuge tube, add 20μl of Proteinase K (500ug/ml), and mix well. Incubate in a 65℃ constant temperature water bath for 30min, or alternatively, in a 37℃ water bath for 12-24h, occasionally shaking the centrifuge tube. Centrifuge at 12000 rpm for 5min in a benchtop centrifuge, and transfer the supernatant to another centrifuge tube.

2. Add 2 volumes of isopropanol, invert to mix. Filamentous precipitate should be visible. Pick it out with a 100ul tip, air dry, and re-dissolve in 200ul TE. (This can be used for PCR reactions, etc.; for further purification, proceed as follows).

3. Add an equal volume of phenol:chloroform:isoamyl alcohol, shake to mix, and centrifuge at 12000 rpm for 5min.

4. Transfer the upper phase to another tube, add an equal volume of chloroform:isoamyl alcohol, shake to mix, and centrifuge at 12000 rpm for 5min.

5. Transfer the upper phase to another tube, add 1/2 volume of 7.5mol/L ammonium acetate and 2 volumes of absolute ethanol. Mix well, precipitate at room temperature for 2min, then centrifuge at 12000 rpm for 10min.

6. Carefully discard the supernatant, invert the centrifuge tube onto absorbent paper to remove any remaining liquid droplets adhering to the tube wall.

7. Wash the precipitate once with 1ml of 70% ethanol, centrifuge at 12000 rpm for 5min.

8. Carefully discard the supernatant, invert the centrifuge tube onto absorbent paper to remove any remaining liquid droplets adhering to the tube wall, and air dry at room temperature.

9. Re-dissolve the precipitate in 200ul TE, then store at 4℃ or -20℃ for later use.

10. Pipette an appropriate amount of sample onto GeneQuant to measure concentration and purity.

IV. Common Issues

1. The experimental materials selected should be fresh, and the processing time should not be too long.

2. Before adding cell lysis buffer, cells must be uniformly dispersed to reduce DNA clump formation.

3. Extracted DNA is difficult to dissolve: impure, containing many impurities; adding too little dissolving solution makes the concentration too high. If the precipitate is too dry, it will also make dissolution difficult.

4. DNA appears smeared during electrophoresis detection: careless operation; contamination with nucleases, etc.

5. Spectrophotometric analysis of DNA shows A280/A260 less than 1.8; impure, containing proteins and other impurities. In this case, add SDS to a final concentration of 0.5%, and repeat steps 2-8.

6. After phenol/chloroform/isoamyl alcohol extraction, the supernatant is too sticky to aspirate: contains high concentration of DNA, the amount of buffer before extraction can be increased or the amount of tissue taken can be reduced.

Experiment 7: Quantification of DNA

I. Experimental Principle

Nucleic acid molecules (DNA or RNA) have a specific ultraviolet absorption peak at 260 nm wavelength due to the conjugated double bonds of purine and pyrimidine rings. Their absorption intensity is directly proportional to the nucleic acid concentration. This physical property provides the basis for determining the concentration of nucleic acid solutions.

1 OD260 corresponds to 50 ug/ml for dsDNA, 33 ug/ml for ssDNA, and 40 ug/ml for ssRNA. This can be used to calculate the concentration of nucleic acid samples.

Ultraviolet spectrophotometry can not only determine the concentration of nucleic acids but also estimate their purity by measuring the ratio of ultraviolet absorption values at 260 nm and 280 nm (A260/A280). The ratio for pure DNA products is 1.8, and for RNA, it is 2.0. If DNA is higher than 1.8, there might be RNA contamination, and if it is lower than 1.8, there is protein contamination.

For very dilute nucleic acid solutions, fluorometry can be used. DNA itself does not produce fluorescence, but after the fluorescent dye ethidium bromide (EB) intercalates between the base pairs of DNA and binds to it, the DNA sample can produce orange-red fluorescence under ultraviolet light excitation. The fluorescence intensity is proportional to the amount of bound EB, and the amount of bound EB is proportional to the nucleic acid molecule length and content. By using a series of known DNA solutions of different concentrations as standard controls, the concentration of the tested DNA solution can be compared. The sensitivity can reach 1-5ng. Since it is based on visual observation, it is an estimation level. This method is economical and simple, but its accuracy is lower.

II. Instruments and Reagents

1. Instruments: Amersham GeneQantTM Pro UV-Vis spectrophotometer, agarose gel electrophoresis equipment.

2. Reagents and Preparation:

5× Loading Buffer:

Prepare 10 ml

0.5 mol/L EDTA (pH 8.0) 2 ml

10% SDS 50 ul

50% Glycerol 2.5 ml

0.2% Bromophenol Blue 10 mg

0.2% Xylene Cyanol FF 10 mg

Add deionized water to 10 ml

Other reagents: 0.1×TE, DNA standard solution, EB stock solution (10 mg/ml).

III. Experimental Procedure

1. Ultraviolet Spectrophotometry

(1) Dilute the DNA sample to be tested with 0.1×TE buffer at a 1:20 ratio or an appropriate multiple.

(2) Turn on the instrument; it will automatically check the optical path and analysis software. When "instrument Ready" appears on the display, enter the nucleic acid measurement window.

(3) Zero adjustment. First, inject 0.1×TE buffer into the sample cuvette, place it in the sample holder, and close the cover. Click the "set ref" button, and the instrument will automatically calibrate the zero point. Remove the sample cuvette from the sample holder and replace it with the sample to be tested.

(4) Aspirate 70 ul of the diluted DNA sample into a quartz cuvette, place it in the sample holder, and close the cover. If the sample volume is very small, a 5-7 ul quartz cuvette can be used. Click the "enter" button, and the instrument will enter the analysis state. The window will simultaneously display the optical density (OD values) at 260 nm and 280 nm, the 260/280 nm and 280/260 nm ratios, and the concentration of the DNA sample, etc.

(5) Open the cover, remove the quartz cuvette, aspirate the sample solution, clean the quartz cuvette with high-purity water, air dry, and then add the next sample to be tested.

(6) DNA purity: reflected by the OD260/OD280 ratio. When the OD260/OD280 ratio is <1.8, it indicates that the sample contains impurities such as protein or phenol. Re-extraction with equilibrated phenol/chloroform-isoamyl alcohol can be used to remove protein, or ether extraction can be used to remove residual phenol, followed by anhydrous ethanol precipitation and resuspension in TE before re-measurement. When the OD260/OD280 ratio is >2.0, it indicates RNA contamination in the sample, and the sample can be treated with RNase to remove RNA.

2. EB Fluorescence Analysis Method

(1) Preparation of agarose gel.

(2) Sample preparation. Perform 1:2 serial dilutions of the DNA sample to be tested, and prepare a DNA standard solution as a control.

(3) Loading. Load the diluted sample after mixing uniformly with loading buffer at a 1:5 ratio.

(4) Electrophoresis. Perform constant voltage electrophoresis at 100V until the bromophenol blue indicator migrates to 3/4 of the gel, then stop electrophoresis.

(5) Remove the gel, incubate in EB fluorescent dye for 20 min, then rinse thoroughly with clear water, and finally perform UV detection. The highest dilution visible to the naked eye contains approximately 80 ng of DNA. This method can also be used to understand cases where nucleic acid samples are severely contaminated with substances that interfere with nucleic acid UV absorption.

IV. Common Issues

1. UV spectrophotometry cannot distinguish the configurations of DNA molecules, such as supercoiled, open-circular, and linear forms of plasmid DNA molecules, nor can it distinguish between chromosomal DNA and RNA. When measuring absorbance at A260, it is difficult to exclude the influence of factors such as RNA, chromosomal DNA, and the hyperchromic effect of DNA denaturation, so the measured data are often higher than the actual concentration.

2. The OD320 value is the background; if the salt concentration of the solution is higher, the OD320 will also be higher.

3. The quartz sample cuvettes used for sample measurement are relatively expensive. Be careful not to break them, and avoid touching the transparent optical surfaces when holding the cuvette to prevent interference with the measurement.

Experiment 8: PCR Gene Amplification

I. Experimental Principle

Polymerase Chain Reaction (PCR) is an in vitro nucleic acid amplification system. Its principle is similar to the natural replication process of DNA molecules. It involves denaturing, annealing, and extending a DNA fragment to be amplified with two complementary oligonucleotide primers on both sides for several cycles, resulting in DNA amplification by 2n fold. This technique has become an essential tool in molecular biology for DNA cloning and gene analysis.

The PCR working procedure is essentially an enzyme-catalyzed synthesis reaction dependent on DNA polymerase in the presence of template DNA, a pair of oligonucleotide primers with known sequences, and four deoxyribonucleotides. The specificity of amplification depends on the binding of primers to the template DNA. The entire amplification process consists of three steps: ① Denaturation, heating to break the hydrogen bonds between the double strands of template DNA to form two single strands; ② Annealing, rapidly lowering the temperature allows the template DNA primers to bind complementarily according to the base pairing principle. Binding also occurs between the two template strands, but due to the high concentration and simple structure of the primers, the primary binding occurs between the primer and the template; ③ Extension, in the presence of DNA polymerase and magnesium ions, single nucleotides are added starting from the 3' end of the primer, forming a new DNA strand complementary to the template strand. Completing these three steps constitutes one cycle. After each cycle, the amount of DNA in the sample doubles, and the newly formed DNA strand becomes the template for the next round of cycling. After 25-40 cycles, DNA can be amplified 10^6 to 10^9 times. The principle of PCR is illustrated below:

II. Instruments and Reagents

1. Instruments: PCR machine, benchtop centrifuge, pipettes and tips, PCR thin-walled tubes, electrophoresis apparatus, etc.

2. Reagents:

10X PCR Buffer Preparation (partially supplied with Taq enzyme):

250-500 mmol/L KCl

100-500 mmol/L Tris-Cl(pH8.4)

15-20 mmol/L MgCl2

0.5% Tween-20

1mg/L BSA

Other reagents: Template DNA, dNTP Mixture, Taq polymerase, upstream and downstream oligonucleotide primers, agarose gel, etc.

III. Operating Procedures

1. PCR System (40ul):

4ul 10XPCR Buffer

4ul 25mM MgCl2 (depends on the PCR buffer from different companies)

Xul Template DNA ≥50ng

1ul Upstream Primer (50-100ng)

1ul Downstream Primer (50-100ng)

1ul dNTP Mixture (final concentration 20-200uM)

0.4 ul Taq Polymerase

Add ddH2O to 40ul

Depending on whether the PCR machine has a hot lid, do not add or add paraffin oil.

2. Add the above reagents sequentially to the PCR thin-walled tubes. After adding the samples, gently flick to mix, then centrifuge at 6000 rpm for 15 sec to collect reaction components at the bottom of the tube.

3. PCR Reaction Thermal Cycling Program Settings:

(1) 95℃ 300s Denaturation

(2) 94℃ 45s Denaturation

(3) 55℃ 45s Annealing (annealing temperature may vary depending on the primers)

(4) 72℃ 45s Extension (can extend 1kp per minute)

Repeat steps 2-4 for a total of 30 cycles

(5) 72℃ 600s Extension

After the reaction, briefly centrifuge, take a small amount (10ul) for analysis, and place the rest

at 4℃ for storage.

4. Agarose gel electrophoresis detection of PCR product.

IV. Precautions:

1. The actual amount of each component added to the PCR system should be calculated based on the final concentration selected by the experimenter and the concentration of the stock solution.

2. Be diligent when adding samples. Insert the tip vertically into the reagent tube. Change tips after each addition, and mark the samples already added to prevent incorrect or omitted additions and to avoid contamination.

3. Taq polymerase (kept on ice) should be added last to minimize exposure to room temperature. When adding the enzyme, do not insert the tip too deeply, and do not add excessive enzyme.

V. Main Factors Affecting PCR

1. Template DNA

Both single-stranded and double-stranded DNA can serve as PCR templates. DNA samples should be as pure as possible, free from contamination by nucleases, proteolytic enzymes, etc. The amount of template depends on the specific experiment; generally, 100ul of reaction solution contains 10^5-10^6 target molecules. Template denaturation must be thorough during the PCR reaction.

2. PCR Primers

The success of PCR primer design is one of the most critical factors in obtaining high-quality PCR products. When designing and applying, the following important aspects need attention:

(1) The length of PCR primers is about 18-30 nucleotides, and the G+C content between paired primers should be similar, so that they bind to their complementary sequences at similar temperatures. G+C content should be between 45% and 55%. The base distribution is random, and continuous occurrence of more than 4 single bases should be avoided, especially avoiding 3 consecutive Gs or Cs at the 3' end, otherwise the primers will mis-prime in G+C rich sequence regions. The 3' end bases of primers are best chosen from A, G, C, avoiding T as much as possible, especially avoiding more than 2 consecutive Ts.

(2) Generally, for PCR products ≤ 500bp, the primer length should be chosen as 16-18bp; for PCR products ≥ 5kb, a primer length of about 24bp is preferable.

(3) Self-complementarity of primer molecules should be avoided, otherwise hairpin-like secondary structures will form. The 3' end sequences of two PCR primers should not have significant complementarity with each other to avoid forming primer dimers.

(4) The melting temperature (Tm value) of the primers should be appropriate. The Tm value is related to the base composition and length of the primers; the GC/AT content of PCR primers should be comparable to or slightly higher than the template DNA to be amplified. Generally, a melting temperature greater than 55℃ is preferred.

(5) The 3' end of the primer must be strictly complementary to the template, while the requirements for the 5' end can be more flexible.

(6) Currently, computer software is widely used in primer selection to search for relevant gene sequences from EMBL or Genebank, and to analyze and evaluate the designed primers in terms of primer dimer formation, self-complementarity, and specificity.

(7) Primers used for subcloning often have restriction endonuclease sites added to their 5' end. To ensure effective enzymatic digestion of the amplified product by the endonuclease, a few extra bases are usually added to the 5' end of the restriction site.

(8) Within a final concentration range of 0.2 to 1.0 umol/L, the yield of primers is basically the same; below 0.2 umol/L, the yield will be affected. However, if the concentration is too high, it is uneconomical and can lead to non-specific amplification and increased primer dimer formation.

3. Thermostable DNA Polymerase (e.g., Taq DNA Polymerase)

The typical dosage is 2.5 U/100uL reaction volume. Excessive enzyme can easily lead to the production of non-specific products. The optimal temperature for this type of enzyme is 75℃, and it retains considerably high activity at lower temperatures, which can easily cause non-specific extension of incompletely matched primer-templates and amplification extension of primer dimers. Therefore, care should be taken to prevent the appearance of non-specific products.

4. dNTPs

The commonly used dNTP concentration for PCR is 50-200umol/L. All four dNTPs should be in equimolar amounts. Concentration

If too low, the reaction speed decreases; if too high, specificity decreases. The optimal dNTP concentration can be determined based on specific experiments.

5. PCR Buffer

Since dNTPs in PCR reactions can bind with Mg2+, affecting the concentration of free Mg2+ in the reaction solution, an appropriate Mg2+ concentration should be determined based on different conditions. Generally, in standard PCR reactions (dNTP concentration 200umol/L), the Mg2+ concentration is about 1.5mmol/L. Mg2+ ion concentration can significantly affect PCR yield and product specificity. High concentrations lead to non-specific amplification, while excessively low concentrations significantly reduce enzyme activity. The use of nuclease-free BSA, Tween-20 (0.05%~0.1%), and 5mmol dithiothreitol (DTT) provides some protective effect on the enzyme.

6. PCR Cycling Temperatures

Initial Denaturation: The initial denaturation time should be longer to ensure complete denaturation. It is generally 94℃ for 3-5min. If denaturation is incomplete, DNA double strands will quickly re-anneal, affecting the PCR reaction. However, if the denaturation temperature is too high (should not exceed 95℃), it will affect Taq enzyme activity.

Denaturation: Generally 94℃ for 30s or 95℃ for 20s.

Primer Annealing: Should be determined based on the base pairing between primer and template in the specific reaction and the purpose of the experiment. It is generally 50-72℃. Increasing the annealing temperature enhances the recognition of incorrectly annealed primers and also reduces the mis-extension of incorrect nucleotides at the 3' end of the primer.

Extension: Generally 72℃ for 60-90s. After the last cycle, it should be held at 72℃ for 5min to ensure the integrity of PCR product synthesis.

7. Plateau Effect and Number of PCR Cycles

In the later stages of PCR gene amplification, due to the accumulation of products, the exponential increase in product will turn into a flat curve, which is known as the plateau effect. Its occurrence is caused by multiple factors such as continuous consumption of PCR reagents, reduced enzyme stability, inhibitory effects of end products, and non-specific products. Therefore, selecting a reasonable number of cycles allows for obtaining a sufficient amount of PCR product without unnecessarily increasing the number of cycles.

8. Operating Environment

Avoid environmental contamination and cross-contamination, such as nuclease contamination, non-target DNA contamination, etc.

Experiment Nine: Agarose Gel Electrophoresis for Separation and Purification of Target DNA

I. Experimental Principle

Separation and purification of DNA restriction fragments are common methods in genetic engineering. During electrophoresis, fragments of different sizes separate and locate at different positions. The target fragment can be excised and recovered using the low melting point agarose recovery method. Many types of low melting point agarose can melt into liquid at 65℃ and solidify into a gel at 30℃. Since the denaturation temperature of double-stranded DNA is higher than 65℃, the gel can be melted without denaturing the DNA, allowing for the recovery of DNA fragments when the gel is in a liquid state. The advantage of this method is that various enzymatic reactions, such as synthesizing isotope probes, performing restriction enzyme digestion, and ligation reactions, can be directly carried out in the melted gel.

II. Equipment and Reagents:

1. Equipment

Electrophoresis apparatus, gel imaging system, pipettors, centrifuge tubes, tips, etc.

2. Reagents:

Low melting point agarose, DNA to be purified, gel extraction kit, deionized water or TE (pH7.6).

III. Operating Procedures:

1. Prepare a 1% agarose gel, cut a slot parallel to the sample well at an appropriate position, and replace it with low melting point agarose gel.

2. Load samples, perform 100V electrophoresis, and observe whether the desired fragment has moved into the low melting point gel.

3. Under UV light, cut the low melting point gel containing DNA, place it in a 1.5 ml centrifuge tube, and melt it in 70°C hot water.

4. Operate according to the gel recovery kit instructions.

12. Take an appropriate amount of purified target DNA for detection in GeneQuant to determine purity and concentration, and store the remaining samples at -20°C.

IV. Precautions:

1. The conical flask, electrophoresis tank, and gel casting tray used for preparing the gel must be thoroughly rinsed first.

Experiment Ten: DNA Recombination

I. Experimental Principle

The ligation of exogenous DNA with vector molecules is DNA recombination, and this recombined DNA is called a recombinant. In vitro DNA ligation and recombination is one of the core technologies in genetic engineering operations, and its essence is an enzymatic reaction process. That is, under certain conditions, DNA ligase catalyzes the interaction between the 5' phosphate end and the 3' hydroxyl end of two double-stranded DNA fragments, forming a phosphodiester bond. There are two commonly used DNA ligases: T4 bacteriophage DNA ligase and E. coli DNA ligase. Among them, T4 bacteriophage DNA ligase has lower substrate requirements, can more effectively ligate blunt ends of DNA, and is more widely used.

T4 bacteriophage DNA ligase catalyzes DNA ligation in 3 steps: ① First, ATP and T4 bacteriophage DNA ligase form an enzyme-ATP complex through the phosphate of ATP and the amino group of lysine in the ligase. ② The activated AMP is then transferred from the lysine residue to the 5' phosphate group of one DNA strand, forming a phospho-phosphate bond. ③ The hydroxyl group at the 3' end of the DNA strand is activated, replacing ATP to form a phosphodiester bond with the 5' phosphate group of the DNA, releasing AMP, and completing the ligation between DNA molecules. T4 bacteriophage DNA ligase requires magnesium ions and ATP as cofactors, and the ligation reaction proceeds under specific temperature and pH conditions.

II. Equipment and Reagents:

1. Instruments:

Benchtop centrifuge, pipettes, tips, constant temperature water bath, etc.

2. Reagents:

T4 DNA ligase, 10× T4 DNA ligase buffer, vector DNA, exogenous DNA.

III. Operating Procedures

1. Ligation of exogenous DNA fragments and vector DNA

Take one sterile Ep tube and add the components in the following order using a micropipette.

10× T4 DNA Ligase Buffer 2.5 ul

Digested DNA fragment *1 approx. 0.3 pmol

Digested vector DNA *2 approx. 0.03 pmol

T4 DNA Ligase 1ul

ddH2O add to 25ul

*1 The molar amount of the DNA fragment should be controlled at 3-10 times that of the vector DNA.

*2 When ligating a vector with a DNA fragment having identical ends, the vector should first be dephosphorylated to prevent self-ligation.

2. Cap the tube, flick the Ep tube gently with your finger several times, and centrifuge for 2s in a benchtop centrifuge to collect the solution.

3. Place the reaction tube at 16°C for overnight reaction (12～16h).

4. Take approximately 25ng of the DNA ligation mixture for agarose gel electrophoresis to identify the ligation result, using unligated plasmid DNA fragments and digested DNA fragments as controls for electrophoresis.

5. Dilute the ligation mixture to 50ul with 0.1× TE, and use 10～20ul to transform E. coli competent cells.

IV. Precautions

1. Ligation products should be promptly used for transformation experiments after identification.

2. Adjust the enzyme dosage according to specific conditions. Blunt-end ligation or adaptor ligation is much slower than sticky-end ligation, and also requires an increased amount of enzyme.

3. Monovalent cations or low concentrations of polyethylene glycol can improve the efficiency of blunt-end ligation.

4. To prevent self-circularization of vector DNA, linear vectors are often treated with alkaline phosphatase to remove the 5' phosphate of the vector, thereby inhibiting self-circularization of DNA fragments.

5. Correctly adjusting the ratio between vector DNA and exogenous DNA helps in obtaining high yields of recombinant products.

V. Related Questions

1. Factors Affecting Ligation Reaction

In addition to factors such as the concentration and ratio of the vector and donor, many other factors affect the ligation reaction, including buffer components, ligation temperature and time, enzyme concentration, DNA concentration, and the base sequence at the ends of the fragments.

(1) Effect of Ligation Buffer

Ligation buffers provided by different companies may vary, but generally contain the following components: 20-100mmol/L Tris-HCl, commonly 50mmol/L, with a pH range of 7.4-7.8, commonly 7.8, to provide an appropriate pH for the ligation system; 10mmol/L MgCl2, which activates the enzymatic reaction; 1-20mmol/L DTT, commonly 10mmol/L, which maintains a reducing environment and stabilizes enzyme activity; 25-50ug/ml BSA, which increases protein concentration to prevent enzyme inactivation due to overly dilute protein concentration. Unlike restriction enzyme buffers, ligase buffers also contain 0.5-4mmol/L ATP, now commonly 1mmol/L, which is essential for the enzymatic reaction.

(2) Effect of pH

Generally, the pH of the buffer is adjusted to 7.4-7.8, with 7.8 being most common. Experiments have shown that if the enzyme activity at pH 7.5-8.0 is defined as 100%, then in an alkaline system (pH 8.3) it is only 65% of the total activity; when the system is acidic (pH 6.9), it is only 40% of the total activity.

(3) Effect of ATP concentration

The concentration of ATP in the ligation buffer is between 0.5-4mmol/L, with 1mmol/L being more commonly used. Studies have found that the optimal ATP concentration is 0.5-1mmol/L, and too high a concentration will inhibit the reaction. For example, 5mmol/L ATP will completely inhibit blunt-end ligation, and 10% of sticky-end ligation is also inhibited. There are also reports that when the ATP concentration is 0.1mmol/L, the self-ligation ratio of dephosphorylated vectors is highest. Since ATP is very easily decomposed, when a ligation reaction fails, besides issues with DNA and enzymes, the ATP factor should also be considered. ATP-containing buffer should be stored at -20℃ and immediately returned to storage after thawing and use. When the volume of ligation buffer is large, it is best to store it in small aliquots to prevent ATP decomposition caused by repeated freezing and thawing. Unlike restriction enzyme buffers, ATP-containing ligation buffers often become ineffective after long-term storage, so you can also prepare an ATP-free buffer yourself (which can be stored for a long time) and add freshly prepared ATP stock solution when needed.

(4) Effect of ligation temperature and time

Because of the hydrogen bonds between the DNA double strands of sticky ends, excessively high temperatures will make hydrogen bonds unstable, but the optimal temperature for ligase is precisely 37℃. To resolve this contradiction, after comprehensive consideration, the ligation temperature is traditionally set at 16℃ for 4-16 hours. Recent experiments have found that for general sticky ends, 20℃ for 30 minutes is sufficient to achieve quite good ligation results. Of course, if time permits, 20℃ for 60 minutes can make the ligation reaction more complete. For blunt ends, there is no need to consider hydrogen bonding issues, and higher temperatures can be used to better optimize enzyme activity.

(5) Effect of enzyme concentration

According to New England Biolabs' definition of T4 DNA ligase, 1U of enzyme can ligate 50% of Hind Ⅲ digested fragments (sticky ends at 0.12umol/L 5' ends, with DNA mass concentration approximately 300ng/ul) in a 20ul system at 16℃ in 30 minutes. The DNA concentration used daily is 10-20 times lower than the enzyme unit definition state, and the amount of enzyme used for blunt-end ligation is 10-100 times higher than for sticky-end ligation. To meet this requirement, manufacturers often provide high-concentration enzymes (hundreds of units/ul), so dilution is required before sticky-end ligation. Except for the portion immediately used for the reaction, which can be diluted with enzyme reaction buffer, all other portions should be diluted with the diluent provided by the manufacturer. The diluent's components are the same as or similar to the enzyme storage buffer, and the enzyme in the diluent can maintain activity for a long time, making it convenient for anytime use.

(6) Effect of DNA concentration

Although some experimental manuals recommend using a DNA concentration of 0.1-1nmol/L, considering that plasmid cloning ultimately requires circularized effective ligation products, the DNA concentration should not be too high, generally not exceeding 20nmol/L. In comparison, cloning processes using bacteriophages and cosmid plasmids as vectors ultimately require linearized ligation products, so the DNA concentration can be higher, at least close to the recommended concentration. Furthermore, when cloning large fragments with large plasmid vectors, and in the ligation reaction of double-digested fragments, the DNA concentration should be further reduced, even down to a few nmol/L for the total DNA concentration. According to another study, the apparent Km value of T4 DNA ligase for DNA ends is 1.5nmol/L, so the DNA concentration during ligation should not be lower than 1nmol/L, which means it should have a terminal concentration of 2nmol/L.

2. Definitions of Three Ligation Enzyme Units

(1) Weiss Unit

The ligase unit was first proposed by Weiss in 1968 as the Weiss unit, now also called the PPi unit. His unit is defined as the amount of enzyme required to transfer 1nmol of 32P from pyrophosphate to an ATP molecule within 20 minutes at 37℃. Most manufacturers still use this unit today.

(2) d(A-T) Circularization Unit

Because the Weiss unit definition tests a different phosphate exchange function of T4 DNA ligase besides its ligation function, and the testing temperature is as high as 30℃, there is a certain gap compared to actual ligation reactions in all aspects. Therefore, in 1970, Modrich and Lehman proposed the d(A-T) circularization unit, which truly measures the ligation function, also known as exonuclease resistance assay. The d(A-T) circularization unit is defined as: converting 100nmol/L of d(A-T) (approximately 2kb long) into an exonuclease-resistant form within 30 minutes at 30℃.

(3) Sticky-End Unit

Compared to the Weiss unit, the d(A-T) circularization unit is closer to reality, but still has some issues. For example, the circularization unit test uses pure AT fragments, which does not match the random arrangement of the four bases in actual ligation. Furthermore, if the ligase connects fragments into multimers but does not circularize them, they may still be cut by exonucleases. In addition, compared to the 16℃ temperature used in most actual ligation reactions, 30℃ still seems too high. To measure enzyme activity under actual ligation conditions, New England Biolabs proposed the most practical sticky-end unit, which is defined as: ligating 50% of λ-HindⅢ digested fragments with a 5' end concentration of 0.12umol/L within 30 minutes at 16℃.

Experiment 11 Extraction of Total RNA from Animal Tissue Cells

I. Experimental Principle

RNA is an intermediate product of gene expression, existing in the cytoplasm and nucleus. Manipulating RNA holds significant importance in molecular biology. Obtaining highly pure and intact RNA is essential for many molecular biology experiments, such as Northern blotting, cDNA synthesis, and in vitro translation, where success largely depends on RNA quality. Since most RNA within cells exists in the form of ribonucleoprotein complexes, high concentrations of protein denaturants are used during RNA extraction to rapidly disrupt cell structures, separate ribonucleoproteins from RNA, and release RNA. Subsequently, treatment with organic solvents like phenol and chloroform, followed by centrifugation, separates RNA from other cellular components, yielding purified total RNA. During the extraction process, it is crucial to inhibit endogenous and exogenous RNase activity to protect RNA molecules from degradation. Therefore, extraction must be performed in an RNase-free environment. RNase inhibitors can be used; for example, DEPC is a strong RNase inhibitor often used to inhibit exogenous RNase activity. Extraction buffers generally contain protein denaturants such as SDS, phenol, chloroform, and guanidine salts, which also inhibit RNase activity and help remove non-nucleic acid components.

This experiment introduces the guanidinium thiocyanate method and TRIzol method for extracting total RNA from animal tissues and identifying it by electrophoresis.

II. Instruments and Reagents

1. Instruments:

Laminar flow hood, high-speed refrigerated centrifuge, electrophoresis apparatus, UV spectrophotometer, gel imaging system, shaker, pipette, tips, Ep tube, glass homogenizer, test tube,

After washing, glassware should be baked at 180℃ for 8h; heat-sensitive instruments (e.g., plastic products) should be soaked in 0.1% DEPC for 2h, baked dry at 70-80℃, autoclaved at 120℃ for 20min, and then baked dry at 70-80℃ before use.

2. Reagents:

(1) Cell lysis solution:

Guanidinium thiocyanate 4mol/L

Sodium citrate (pH7.0) 25mmol/L

Sodium lauroyl sarcosinate 0.5%

β-mercaptoethanol 0.1mol/L

Weigh 0.64g sodium citrate, 0.415g sodium lauroyl sarcosinate, take 0.7ml β-mercaptoethanol, dissolve in Rnase-free distilled water, and make up to 50ml. Then take 33ml of the prepared solution (CBS solution) and 25g guanidinium thiocyanate, mix, and store at 4℃ after complete dissolution for later use.

(2) 10× gel buffer:

Morpholinopropanesulfonic acid (MOPS) (pH7.0) 200mmol/L

NaAc 100mmol/L

EDTA(pH 8.0) 10mmol/L

Store away from light after sterilization by filtration.

(3) 5× denaturing loading buffer:

Water-saturated bromophenol blue 16μl

500mmol/L EDTA(pH 8.0) 80μl

Formaldehyde (37%) 720μl

Glycerol 2ml

Formamide 3084μl

10× gel buffer 4ml

Add RNase-free water to 10ml, can be stored at 4℃ for 3 months.

(4) TRIzol RNA extraction reagent

(5) 2mol sodium acetate

(6) 0.1% DEPC

(7) Equilibrated phenol:chloroform:isoamyl alcohol (25:24:1)

(8) Equilibrated phenol, chloroform

(9) Anhydrous ethanol, 70% ethanol, isopropanol

(10) RNase-free water

III. Operating Procedures

(I) Guanidinium Thiocyanate Method (PLT)

1. Place 0.1-0.2g of fresh animal tissue into a tissue homogenizer, add 1ml of pre-cooled cell lysis solution, and rapidly homogenize in an ice bath for 15-30s to thoroughly grind the tissue. Then aspirate the cell suspension into another test tube.

2. Add 120μl of 2mol sodium acetate (pH4.0) and mix thoroughly.

3. Add 1.2ml phenol:chloroform:isoamyl alcohol, mix thoroughly by shaking for 10s, and incubate on ice for 15min.

4. Transfer the mixture to a 1.5ml Ep tube, centrifuge at 10,000 r/min for 20min at 4℃.

5. Transfer the upper aqueous phase to another Ep tube, add an equal volume of isopropanol, and let stand at -20℃ for 30min to precipitate RNA.

6. Centrifuge at 12,000 r/min for 15min at 4℃.

7. Discard the supernatant, add 400μl of 70% ethanol to wash the RNA precipitate; if the RNA precipitate is suspended, centrifuge at 10,000 r/min for 10min at 4℃.

8. Discard the supernatant, and air dry naturally, but avoid complete drying of the precipitate, otherwise RNA will be difficult to dissolve.

9. Add 100μl RNase-free water to resuspend RNA, or add 1ml anhydrous ethanol and 1/10 volume of 3mol sodium acetate (pH4.0), and store at -70℃.

(II) RNA extraction with TRIzol reagent

1. Place 0.1-0.2g of fresh animal tissue into a tissue homogenizer, add 1ml of pre-cooled Trizol solution into the glass homogenizer, and rapidly homogenize in an ice bath for 15-30s to thoroughly grind the tissue. Then aspirate the cell suspension into another 1.5ml Ep tube, and let stand at room temperature for 5min.

2. Add 200μl of chloroform, shake vigorously for 15s to mix, then let stand at room temperature for 3min.

3. Centrifuge at 10,000 r/min for 15min at 4℃, RNA will be in the aqueous phase.

4. Transfer the upper colorless aqueous phase to another Ep tube, add 500μl of isopropanol, and let stand at room temperature for 10min.

5. Centrifuge at 10,000 r/min for 10min at 4℃.

6. Discard the supernatant, wash the RNA precipitate with 1ml of 75% ethanol, centrifuge at 7500 r/min for 5min at 4℃.

7. Discard the supernatant, and air dry at room temperature for 15min.

8. Add 200μl of DEPC-treated water (nuclease-free water) to the dried precipitate to dissolve it, and store at -20℃ for later use.

(III) RNA detection

1. Detect RNA concentration and purity using a UV spectrophotometer.

The method is the same as in Experiment 4: use DEPC-treated water to calibrate the zero point; dilute the RNA sample with DEPC-treated water; read OD260, OD280 values, and the OD260/OD280 ratio.

2. Agarose Gel Formaldehyde Denaturing Electrophoresis Detection

(1) Prepare the gel (1.2%). Weigh 1.2g agarose, add 72ml DEPC-treated water, and heat to melt. Cool to 60℃, add 10ml of 10× gel buffer and 18ml of formaldehyde (37%) in a fume hood. Mix well, then pour the gel.

(2) Prepare the sample. In a centrifuge tube, mix the RNA sample with 5× denaturing loading buffer at a 4:1 ratio. Incubate at 65℃ for 5-10min, quickly cool on ice for 5min, and centrifuge for a few seconds.

(3) The gel must be pre-electrophoresed for 5min before loading samples. Then, load the samples into the wells. Electrophorese at 5V/cm for 1.5-2h.

(4) End electrophoresis when bromophenol blue migrates to 2/3 to 4/5 of the gel length. Stain the gel in ethidium bromide solution (0.5μg/ml, prepared with 0.1mol/L ammonium acetate) for about 30min.

(5) Observe and analyze using a gel imaging system.

IV. Precautions

1. RNA is a nucleic acid molecule that is highly susceptible to degradation. Therefore, total RNA extraction must be performed in an RNase-free environment, wearing masks and gloves, and using RNase-free reagents, materials, and containers.

2. All solutions should be treated with 0.05%–0.1% DEPC at room temperature overnight, then autoclaved or heated to 70℃ for 1 hour or 60℃ overnight to remove residual DEPC.

3. The chemical reagents used should be new packages, and a dry-baked weighing spoon should be used for weighing. All operations should be carried out in an ice bath; low temperature conditions can reduce RNase activity.

4. Based on the UV absorption OD value of the RNA sample, the RNA concentration can be calculated.

Single-stranded RNA [ssRNA]=40×(OD260-OD310)×Dilution Factor

The OD260/OD280 ratio for pure RNA is usually between 1.7 and 2.0. If the ratio is below 1.7, it indicates contamination with proteins, etc., and phenol/chloroform extraction should be applied. If the ratio is below 2.0, it indicates the presence of salts, guanidine, or sugars, and LiCl can be used to selectively precipitate RNA to remove impurities.

5. After RNA sample electrophoresis, if 28S, 18S, and 5S small molecule RNA bands are visible, it indicates good integrity. If degradation occurs, it may be due to improper operation or RNase contamination. The ratio of 28S to 18S RNA is approximately 2:1, indicating no RNA degradation. If the ratio is reversed, it indicates RNA degradation. If there are bands behind 28S during electrophoresis, it indicates DNA contamination, and purification should be performed after DNase treatment.

Main references:

1. Jin Dongyan et al. (translators). Molecular Cloning: A Laboratory Manual. Second Edition. Beijing: Science Press, 1995

2. Zi Ying et al. (translators). Concise Molecular Biology Laboratory Manual. Science Press,

3. Zhou Junyi (editor). Basic Skills and Strategies in Molecular Biology. Science Press

4. Jiang Bo et al. (editors). Common Experimental Methods in Molecular Biology. Beijing: People's Military Medical Press, 1996.

5. Liu Jinyuan et al. (editors). Molecular Biology Experimental Guide. Beijing: Tsinghua University Press, 2002.