Experimental Guidance

Table of Contents

Experiment 1 Common Instruments and Usage in Molecular Biology Laboratory

Experiment 2 Plasmid DNA Extraction - Alkaline Lysis Method

Experiment 3 Agarose Gel Electrophoresis

Experiment 4 Restriction Endonuclease Digestion and Identification

Experiment 5 Preparation and Transformation of Competent E. coli Cells

Experiment 6 Genomic DNA Extraction from Animal Tissue Cells

Experiment 7 DNA Quantitation

Experiment 8 PCR Gene Amplification

Experiment 9 Agarose Gel Electrophoresis Separation and Purification of Target DNA

Experiment 10 DNA Recombination

Experiment 11 Total RNA Extraction from Animal Tissue Cells

Experiment 1 Common Instruments and Usage in Molecular Biology Laboratory

Facts prove that in today's rapidly developing science, no matter which field of research one is engaged in, to make breakthroughs, in addition to a good theoretical foundation, it is more important to rely on advanced technology, excellent instrumentation, and a good research environment. A standard molecular biology laboratory, in addition to the conventional instruments and equipment of a general biology laboratory, also has some instruments for special purposes. These instruments are generally precise and expensive. The following introduces the usage methods and precautions for these instrumentsulators

I. Refrigerated Centrifuge

Low-temperature separation technology is an indispensable means in molecular biology research. Low-temperature centrifugation is essential for the separation of gene fragments, the precipitation and recovery of enzyme proteins, and the separation and preparation of other biological samples. Therefore, the low-temperature refrigerated centrifuge has become an essential important instrument in molecular biology research. In China, multiple manufacturers produce refrigerated centrifuges. The high-speed refrigerated centrifuge in this laboratory is a floor-standing GL-20G-Ⅱ model (Shanghai Anting). It is equipped with angle rotors: 6×50ml, 12×10ml, and 12×1.5ml. The maximum speed is 20000rpmulators

1. Installation and Debugging

The centrifuge should be placed on a level and sturdy surface, at least 10cm away from walls, in a well-ventilated environment. The surrounding air should be neutral, free of conductive dust, flammable gases, and corrosive gases. The ambient temperature should be between 0 and 30℃, and the relative humidity should be less than 80%. Before the test run, open the lid, rotate the shaft by hand to ensure it moves smoothly and freely without any abnormalities, then attach the required rotor. After the rotor is correctly in place, turn on the power switch, then hold down the door switch, and press the run button. After rotation, immediately stop and observe the direction of the shaft. If it rotates counter-clockwise, it is correct, and the machine can be put into useators

2. Operating Procedure

(1) Plug in the power, and the standby indicator light will illuminate; turn on the power switch, the flashing digital display of the speed control and timing system shows the factory setting for the machine's operating speed, and the digital display of the temperature control system shows the current temperature of the centrifuge chamberators

(2) Set the machine's operating parameters, such as operating temperature, running time, and operating speedators

(3) Place the pre-balanced samples onto the rotor sample rack and close the lidators

(4) Press the run button on the control panel, and the centrifuge will start running. Within the pre-set acceleration time, its speed will increase to the pre-set valueators

(5) Within the pre-set running time (excluding deceleration time), the centrifuge will begin to decelerate, and its speed will drop to zero within the pre-set deceleration timeators

(6) Press the stop button on the control panel, the digital display will show "dedT", and after a few seconds, it will show a flashing speed value. At this point, the machine is ready for the next operationators

3. Precautions

(1) The centrifuge should always be in a horizontal position. The voltage of the external power system must match, and a good ground wire is required. When the machine is not in use, unplug the power cordators

(2) Before starting, check whether the rotor is firmly installed and if any foreign objects have fallen into the chamberators

(3) Samples should be pre-balanced. When centrifuging with centrifuge tubes, both the centrifuge tube and the sample should be balanced simultaneouslyators

(4) When centrifuging volatile or corrosive liquids, use capped centrifuge tubes and ensure no liquid leaks out to prevent corrosion of the chamber or accidentsators

(5) Do not arbitrarily change the machine's operating parameters other than operating temperature, running speed, and running time, to avoid affecting machine performance. The set speed must not exceed the maximum speed to ensure safe operation of the machineators

(6) If "0.00" or other numbers appear during use and the machine does not operate, shut down and disconnect the power. After 10 seconds, restart the machine. Once the set speed is displayed, press the run button, and the machine will operate as usualators

(7) Do not open the lid during machine operation or before the rotor has come to a complete stop, to avoid accidentsators

(8) After each operation, record the usage and regularly inspect the machine's performanceators

II. Electrophoresis Apparatus

Electrophoresis technology is an indispensable and important tool in molecular biology research. Electrophoresis is generally divided into two main categories: free boundary electrophoresis and zone electrophoresis. Free boundary electrophoresis does not require a support medium, such as isotachophoresis, density gradient electrophoresis, and microelectrophoresis; these types of electrophoresis are rarely used now. Zone electrophoresis requires various types of substances as support media. Commonly used support media include filter paper, cellulose acetate membrane, non-gel supports, gel supports, and silica gel-G thin layer. Agarose gel electrophoresis is the most commonly used in molecular biology experiments. Electrophoresis can be used for qualitative or quantitative analysis of different substances, or for component analysis of certain mixtures, or for the extraction and preparation of individual components. The following introduces the usage method, taking the DYY-12 Computerized Triple-Constant Multipurpose Electrophoresis Apparatus (Beijing Liuyi Instrument Factory) as an exampleators

1. Usage Method

(1) Press the power switch. After the display screen shows "Welcome to use DYY-12 Computerized Triple-Constant Multipurpose Electrophoresis Apparatus..." and similar text, the system initializes simultaneously, beeps 4 times, and common settings are applied. The screen then switches to the parameter setting state, as shown in Figure 1ators

U: 0V U= 100V | Mode： STD

I: 0mA I = 50mA |

P: 0W P= 50W |

T: 00:00 T= 01:00 |

Where: The capital letters U: I: P: T: on the left are the actual values during electrophoresis; the middle section displays the program's constant settings (preset values). Mode: STD (Standard); TIME (Timed); VH (Volt-Hour); STEP (Step-by-step)

(2) Set up the work program. Use the keyboard to input a new work program. For example, if the required working voltage U=1000V, current I is limited to 200mA, power W is limited to 100W, and time T is 3 hours and 20 minutes, with automatic output shutdown at the set time. The operating steps are as follows:

①Press the "Mode" key to change the working mode from Standard (STD) to Timed (TIME).

mode. Each press of the mode key changes the working mode in the following sequence: STD®TIME®VH®STEP®STD.

②First set the voltage U. Press the "Select" key to highlight it, then use the numeric keys to set the parameter value. Press 1000, and the voltage setting is complete.

③Set the current I. Press the "Select" key to highlight I, then input the number 200.

④Set the power P. Press the "Select" key to highlight P, then input the number 100.

⑤Set the time T. Press the "Select" key to highlight T, then input the number 320. If an incorrect input is made, you can press the "Clear" key and re-enter.

⑥After confirming that all parameters are correct, press the "Start" key to initiate the electrophoresis instrument output program. "Start!" will be displayed in the status bar on the screen, accompanied by 4 beeps, reminding the operator that the electrophoresis instrument will output high voltage, so pay attention to safety. The output voltage will then gradually increase to the set value. Simultaneously, "Run" will be displayed in the status bar, along with two continuously flashing high-voltage symbols, indicating that voltage is being output from the port. At the bottom of the status bar, the actual working time (accurate to seconds) is displayed.

⑦Each time the output is started, the instrument automatically saves the current set values into the "MO" storage unit. When needed later, you can press the "Read" key, then the "0" key, and then the "Confirm" key to retrieve and execute the previously set work program.

⑧When electrophoresis ends, the instrument displays: "END" and continuously beeps as a reminder. Press any key at this point to stop the beeping.

2. Precautions

(1) The valid input range for the three parameters U, I, and P is: U: 5～3000V; I: 4～400mA; P: 4～400W.

(2) Generally, when "No Load!" appears, first turn off the instrument and check for any poor contact between the electrode wires and the electrophoresis tank. A multimeter's ohm range can be used to measure segment by segment.

(3) If multiple electrophoresis tanks are connected to the output, the current value displayed by the instrument is the sum of the currents in each tank. In this case, stable voltage output should be selected to minimize mutual influence between the tanks.

(4) Pay attention to keeping the instrument clean and do not block the air inlet channel at the back of the instrument. It is strictly forbidden to place the electrophoresis tank on top of the instrument to avoid buffer solution spilling into the instrument.

(5) The output voltage of this instrument is high; avoid contact with the output circuit and the inside of the electrophoresis tank during use to prevent danger.

(6) If the instrument is not used for a long time, it should be stored in a dry, ventilated, and clean environment.

III. Analytical Balance

Analytical balances are indispensable and important instruments in quantitative analysis. A thorough understanding of instrument performance and proficient mastery of their use are guarantees for obtaining reliable analytical results. There are many types of analytical balances, including ordinary analytical balances, semi-automatic/fully automatic electro-optical analytical balances, and electronic analytical balances. Currently, the laboratory commonly uses electronic analytical balances with a higher degree of automation. Below, we will introduce the performance and usage methods of the electronic analytical balances PL203/01 and AL104/01 (METTLER-TOLEDO). PL203/01 Precision Electronic Balance: maximum weighing capacity 210g; actual readability 0.001g; AL104/01 High-Resolution Electronic Analytical Balance: maximum weighing capacity 110g; actual readability 0.0001g

1. How to Use

(1) Check and adjust the balance to a horizontal position. Check whether the power supply voltage matches (use the configured voltage regulator), and power on the balance for preheating to the required time of 30min as per instrument requirements.

(2) Turn on the balance switch "on", and the balance will automatically perform sensitivity and zero point adjustment. Once all fields on the display briefly light up and the balance returns to zero, formal weighing can begin.

(3) When weighing, place a clean weighing bottle or weighing paper on the weighing pan, close the side door, and the balance will display the weight. Click the "®O/T¬" key to automatically re-zero, then gradually add the substance to be weighed until the desired weight is reached.

(4) After weighing, promptly remove the weighing bottle (paper), close the side door, cut off the power, and register the usage.

2. Precautions

(1) The balance should be placed on a firm, stable cement or wooden table. The room should be clean and dry, and direct sunlight on the balance should be avoided.

(2) When weighing, substances should be placed and removed through the side door. When taking readings, the chamber door should be closed to prevent air movement from causing the balance to swing.

(3) Volatile, corrosive, strong acid, and strong alkali substances should be weighed in a covered weighing bottle to prevent corrosion of the balance.

(4) If the electronic analytical balance is not used for a long time, it should be powered on for preheating regularly, once a week, for 2h each time, to ensure the instrument remains in good working condition.

IV. Spectrophotometer

Different substances absorb incident light of different wavelengths to varying degrees, thus forming characteristic absorption spectra. Based on this principle, colorimetry can be used for qualitative or quantitative analysis of certain colored substances. However, colorimetry is limited to the visible light region and has low accuracy, which is far from meeting the requirements for high-precision trace analysis. With the continuous development of science and technology, analytical instruments are constantly being updated, and the concept of spectrophotometry has been introduced, followed by the application of spectrophotometers. A spectrophotometer consists of a light source, monochromator, absorption cell, detector, and display screen. It is not only suitable for the visible light region but also extends to the ultraviolet and infrared regions. Optical Density (OD) is one of the indicators for quantifying solutes in many solutions, determined by measuring the absorption value of a certain solution to the monochromatic light produced. In molecular biology experiments, UV spectrophotometers are commonly used for nucleic acid solution quantification and preliminary purity assessment. The following describes the use of the UV-Vis spectrophotometer GeneQantTM Pro (Amersham). In addition to detecting nucleic acid sample concentrations, this instrument can also determine protein concentrations and cell culture media concentrations. It can detect samples as low as a few microliters (70µl and 5～7µl). Samples do not require dilution and can be fully recovered after measurement.

Method of Use

(1) Turn on the power switch (ON) and wait a few seconds. A series of data, such as the instrument model and current date, will appear on the display. These data can be reset. When “instrument Ready” appears, you can proceed to the next step.

(2) There are many function keys on the instrument panel, including detection for base sep, Tm, DNA, RNA, oligo, Protein595assay, Protein280 meas, cell culture, etc. For example, to detect DNA, press the DNA key to enter the DNA detection program. On the display screen:

Pathlengh 10mm

Units µg/µl

Use 320nm NO

Dilution Faotor 1

Insert reference,

The above are the instrument's preset reference data. If you press the “enter” key and the “select” key, you can reset the above parameters.

(3) Take a quartz sample cuvette of 70µl or 5～7µl, the capacity depends on the need. First, use a pipette to draw high-purity water into the sample cuvette, then place it into the sample slot on the instrument. When inserting, ensure the optical surface of the sample cuvette faces forward.

(4) Press the “set ref” key to perform a blank test. A series of “0.000” data will appear on the display, prompting to insert the sample “Insert sample”. Remove the sample cuvette, blot dry the water, and after slight drying, likewise draw in the sample to be tested and place it into the sample slot for measurement.

(5) After a sample measurement is complete, press the “stop” key to return to “Instrument Ready”.

(6) Remove the sample cuvette, aspirate the sample, then wash several times with high-purity water and air dry naturally. Since the sample cuvette is very expensive, operate with care. Do not touch the optical surface of the sample cuvette with your fingers. Wash promptly after use with warm water or dilute hydrochloric acid, ethanol, or even chromic acid cleaning solution (do not soak in concentrated acid for more than 15 minutes). The surface should only be wiped clean with a soft lint-free cloth or lens tissue.

V. Digital pH Meter

A pH meter is an essential instrument for preparing solutions in the laboratory. The pH meter in this laboratory is a desktop microcomputer pH meter and thermometer (Hanna), with a pH measurement range of 0.00～14.00pH; and a temperature range of 0.0～100.0℃;

1. How to Use

(1) Connect the pH electrode and temperature probe to the main unit, and connect the main unit to the power supply.

(2) Remove the electrode protective cap. If crystalline salts appear, this is a common phenomenon for electrodes and will disappear after immersion in water. If the membrane glass or dialysis membrane dries out, it can be soaked in HI170300 electrode storage solution for 1 hour.

(3) pH calibration. Immerse the pH electrode and temperature probe in the selected standard buffer solution for 4cm (recommended pH 6.86, 7.01). The buffer value can be adjusted via the “D℃” or “Ñ℃” keys. Press the “CAL” key, and the instrument will display “CAL” and “BUF” symbols along with “7.01” data. When the reading is unstable, the screen will display “NOTREADY”; when the reading is stable, the screen will display “READY” and “CFM”. Press the “CFM” key to confirm the calibration value. After confirming the first calibration point, immerse the pH electrode and temperature probe in the standard buffer solution for 4cm (recommended pH 4.01, 9.18, 10.01); then press the “CAL” key again, and the instrument will display “CAL” and “BUF” symbols along with “4.01” data. Press the “CFM” key to confirm the calibration value.

(4) pH measurement. After calibration is complete, the instrument automatically enters pH measurement mode. Immerse the electrode and temperature probe in the solution to be tested for about 4cm, and wait a few minutes for the electrode reading to stabilize.

2. Precautions

(1) Since the pH211 pH meter has a built-in rechargeable battery, when using it after purchase or a long period of storage, after powering on, calibrating, and completing measurements, the power supply can remain plugged into the power socket. Simply turn off the switch. This ensures the battery charges and the calibration values are saved, allowing for accurate measurements without recalibration next time.

(2) Do not soak the electrode in distilled water, deionized water, or pure water. If the reading deviation is too large (±1pH), it is due to lack of calibration or a dry electrode. To prevent electrode damage, remove the pH electrode from the solution before turning off the power. When the instrument is off, the electrode should be separated from the machine before immersing it in electrode storage solution.

(3) If the instrument has measured several different sample solutions, please clean it with tap water, or clean the electrode with the sample to be measured before inserting the sample solution.

(4) Temperature affects pH readings. To measure accurate pH values, automatic temperature compensation should be performed within a suitable range. Immerse the HI7669/2W temperature probe in the sample, close to the electrode, and wait a few minutes. If the temperature of the solution to be measured is known or the measurement is performed at the same temperature, only manual compensation is needed, and the temperature probe does not need to be connected. The screen will display the temperature reading with a flashing ℃ signal. Temperature can be adjusted via “Ñ℃” or “D℃”.

VI. PCR Instrument

PCR instrument, also known as DNA thermal cycler or gene amplification instrument, enables a pair of oligonucleotide primers to bind to the target sequences on both sides of the positive and negative DNA strands, thereby enzymatically synthesizing millions of copies of target DNA fragments. Each cycle involves three processes at three different temperatures: DNA denaturation, primer annealing, and DNA polymerase-catalyzed extension reaction.

PCR instruments are mainly used in many fields such as basic research and applied research, including gene analysis, sequence analysis, evolutionary analysis, clinical diagnosis, and forensic medicine. With the continuous development of molecular biology, PCR amplification technology has also been widely popularized and applied. Therefore, it is essential to understand the performance of PCR instruments, master the correct operating procedures, and be aware of precautions during use.

Here are the operating instructions and precautions for the PCR amplification instrument (Tl Thermocycler).

1. How to Use

(1) Turn on the power switch, and the instrument enters the ready state; the current instrument temperature and the lid (Lid) temperature are displayed on the screen. If the instrument is running, you must press the “ B ” button (start/stop) to exit the operation and return to the ready state.

(2) Write program segments. In the ready state, press the “ C ” button (programs) to enter the programming state, select a program number, then press the “enter” button to enter the next program; after pressing the “A”(list) button, select a file number (empty program); then press the “enter” button to enter the next program; press the “ABC” button to enter the next program, use the up, down, left, and right arrow keys to select a letter (A, B, C, D, …), then press the “enter” button to enter the next program; press the “name ok” button to complete file naming.

(3) Set the lid temperature. “lid temp： ℃”. The lid temperature is generally 10℃ higher than the denaturation temperature. For example, if the denaturation temperature is 95℃, the lid temperature should be set to 105℃. After the lid preheats, press the “enter” button to proceed to the next program.

(4) Set parameters such as denaturation temperature, denaturation time, extension temperature, extension time, annealing temperature, annealing time, and total number of cycles. From the first step, press the “enter” button sequentially to enter, and use the four shift keys to move the setting position. Set the temperature first, then the time. After all parameters are set, press the “pgm ok” button, and the set program will be automatically saved.

(5) Run the program. Check if the settings are correct. Press the “start” button, select the set program, and start running. The display screen shows the system's operating status. Press “Stop” to stop the operation.

VII. Gel Imaging System

This system is a fully automatic computer-controlled image analysis system, primarily used for capturing images of nucleic acid agarose electrophoresis gels and protein acrylamide electrophoresis gels. After comparison with markers, the molecular weight of samples can be accurately calculated, and nucleic acid electrophoresis can also be precisely quantified. It is an important detection tool for molecular biology and biochemical protein experiments. This system includes a darkroom, UV transilluminator, camera, zoom lens, computer, and professional gel image acquisition and analysis software (version 3.3). The software provides quantitative analysis of electrophoresis gels, spot measurement, PCR density, etc. In addition, it also provides functions such as image acquisition, annotation, printing, sending data and images to Word, Excel, and image processing.

1. Usage Method

(1) Turn on the computer, launch the gel analysis software, and enter the user interface.

(2) Turn on the power switch of the darkroom device for gel image acquisition, place the gel, turn on the UV light source or white light source, connect the acquisition device to the acquisition card on the computer, then select “Image Acquisition” from the “File” menu or the “Image Acquisition” button on the toolbar. The image window will appear with options such as “Start Acquisition”, “Stop Acquisition”, “Acquire Image”, etc. If the image is not clear, adjust the zoom lens on the darkroom to make it clear. Images can be saved directly or processed via “Image Processing” for rotation, cropping, filtering, contrast adjustment, and so on.

(3) For the imported electrophoresis gel image, the electrophoresis gel analysis system can be launched. Select “Electrophoresis Gel” from the “Function Selection” menu or use the “Electrophoresis Gel” tool on the toolbar. The lane analysis toolbar will appear, and a red rectangular box will appear in the “Image Display” sub-window. You can also enter the “Band Analysis” system to number the bands and compare two or more bands.

(4) After image acquisition is complete, turn off the darkroom power switch, remove the gel from the darkroom, clean the glass plate, and let it dry.

VIII. Air Thermostatic Shaker

The shaker (oscillator) is standard laboratory equipment. The SHK-99-Ⅱ type desktop air thermostatic shaker adopts a microcomputer control system, with a temperature range of room temperature +5℃ to 60℃, accuracy ±0.5℃; time range of 1 minute to 99 hours 59 minutes; and rotation speed range of 60 RPM to 250 RPM.

1. Usage Method

(1) Data for each segment on the LED screen:

RPM Temp Time

1 000 00.0 00:00

On the screen, “1” indicates the first segment; if it's the second segment, it will be “2”. “Temp” indicates temperature, “RPM” indicates revolutions per minute, and “Time” indicates duration. If not set, it can accumulate time automatically and run continuously. The values of “Temp”, “RPM”, and “Time” can be modified at any time and run according to the new values.

(2) Working program settings. Press the “F1” button to enter the setting state. You can switch between four sections: speed, temperature, time, and operation. In each section, press the “F2” button to input data. After inputting the tens digit, input the units digit, press the “F2” button, then press the “F2” button again to reach the decimal place, and then press the “F2” button again to return to the tens digit, repeating this cycle.

(3) Press the “F1” button again to return to the running state. Press the “Start” button, the motor will start running, and the timer will begin.

(4) Press the “End” button to terminate system operation.

2. Precautions

(1) Turn on the power. The system will start self-checking. Wait for the “WELCOME” message to appear on the LCD screen, indicating that the self-check is complete, and you can proceed to step 2 for parameter settings. If the screen displays: a. “TEMP ERROR”, it indicates an error in the temperature detection circuit; b. “MOTOR ERROR”, it indicates an error in the motor section.

(2) During operation, you can open the lid of the chamber; this will stop the motor and pause the timer. Close the lid to resume operation.

(3) When work is complete, turn off the power. After shutting down, wait for a period before turning it on again.

IX. Water Purification Device

Molecular biology experiments have increasingly high requirements for water purity. Ordinary distilled water often fails to meet experimental requirements, and most experiments require a second distillation (double-distilled water). This can remove most organic impurities from the water, but it takes a long time to produce, and there are still many inorganic impurities. Many experiments also require deionized water, which needs to be treated with cation and anion exchange resins. Currently, molecular biology laboratories use high-quality ultrapure water. For example, the ultrapure water produced by Milli-Q ultrapure water manufacturing systems from Millipore Corporation in the United States is suitable for many scientific fields, such as molecular cloning, various chromatographic analyses, amino acid analysis, DNA sequencing, enzyme reactions, tissue and cell culture, and other experiments. Below, we introduce the performance and usage of the RO-MB wall-mounted reverse osmosis high-purity water machine. The RO-MB reverse osmosis high-purity water machine (Hangzhou Yongjieda) produces water at 10L/H (at 25℃); water quality: RO pure water: <10μs/cm, high-purity water: >15MΩ.cm; it can use tap water to produce ordinary laboratory water and high-purity water, simultaneously meeting different water quality requirements. The online conductivity meter continuously monitors the produced water quality, ensuring its quality.

1. How to Use

(1) Preparation: First, check if the raw water pipeline and wastewater pipeline connected to the pure water machine are properly connected, and open the raw water valve. Check if the power supply is normal and if the button is in the off state (button popped out). Insert the power plug into a 220V power socket with a ground wire.

(2) Startup: Press the power switch, then the pump switch in sequence. The pure water machine starts producing water, while wastewater is discharged, indicator lights turn on, and it enters automatic operation.

(3) Obtaining pure water: If the RO pure water or high-purity water outlet valve is opened, pure water of the corresponding quality can be obtained. When the high-purity water outlet valve is opened, the measuring instrument displays the conductivity or resistivity of the high-purity water. To obtain high-quality high-purity water, it is recommended to discard the first cup of water before collecting fresh water.

(4) Shutdown: When not in use, turn off the individual buttons to stop the machine. The tap water inlet valve does not need to be closed, as the internal water inlet solenoid valve of the machine has already automatically cut off the water supply. As long as the pipeline valves do not leak, the machine can also remain in automatic standby mode.

2. Precautions

(1) The normal operating ambient temperature for the pure water machine is 15-35℃. Water production will decrease as the temperature drops, and the maintenance temperature in winter should not be lower than 5℃.

(2) The pre-treatment filter cartridge is generally replaced every three to six months. Its actual service life is related to tap water quality, total filtration volume, etc.

(3) The mixed-bed filter cartridge produces approximately 1-3 tons of high-purity water and should be replaced every two to four months. Its actual service life is related to tap water quality, salt content in the water, total water consumption, etc. If the equipment is out of service for more than 2 days, regular flushing and maintenance must be performed. It is advisable to turn on the machine for 30 minutes for flushing once a day in summer, and once every 2-3 days in winter.

(4) During use, if you find that the pump frequently and regularly starts for a few seconds and then stops every few minutes after shutdown, this is caused by pipeline leakage and requires immediate opening of the casing to resolve. If it starts for a few seconds and then stops every half an hour or more, it is a normal phenomenon, which is beneficial for flushing and protecting the reverse osmosis membrane elements, preventing microbial growth and reducing dirt accumulation, especially during hot seasons.

X. Disinfection Equipment

Reagents, glassware, and experimental tools used for bacterial and cell culture and nucleic acid-related experiments should be strictly sterilized. Some experiments also require no nuclease contamination, so experimental instruments, reagents, etc., should be high-pressure sterilized. For bacterial strains that have undergone recombination with imported DNA molecules, strict high-pressure sterilization and inactivation treatment must be performed after operation.

Large quantities of experimental items, reagents, culture media, etc., can be disinfected using large sterilizers. Some reagents that cannot withstand high-pressure or high-temperature sterilization can be sterilized by filter membranes. Glassware can be disinfected by UV irradiation, 75% ethanol, or soaking in 0.1% sodium dodecyl sulfate (SDS). All cell culture and bacterial culture operations should be performed in a UV-sterilized ultra-clean workbench.

Below, we introduce the usage of the vertical automatic pressure steam sterilizer (LDZX-40BI).

1. Usage Method

(1) Opening the lid: Turn the handwheel to lift the lid off the sealing ring.

(2) Powering on: Connect the automatic water inlet device. Turn on the power, press the power switch on the control panel to ON. If the low water level (LOW) red light is on, the evaporation pot is in a water cut-off state; if the water shortage (LACK) yellow light is on, power has been supplied normally.

(3) Adding water: Open the water source. When the water level reaches the low level, the low water level red light and water shortage yellow light on the control panel will turn off. The heating (1-green light) will turn on. Continue adding water until the high water level (HIGH) green light turns on, and water addition will stop automatically.

(4) Stacking items: Sterilized items that need to be wrapped should preferably have a volume not exceeding 200mm×100mm×100mm. Leave gaps between packages and stack them inside a metal frame, which facilitates steam penetration and improves sterilization effectiveness.

(5) Sealing the autoclave: Push the crossbeam into the column, rotate the handwheel to press down the lid, and fully tighten it.

(6) Setting temperature: After powering on, the digital display window lights up. The upper layer is red, displaying the temperature; the lower layer is green, for setting temperature and time. First, press the 'Confirm' button on the control panel; the green digital display will flash, entering the temperature setting mode. Press the 'Shift' button once, and the corresponding position indicated will flash. Shift (single-item cycle) according to the desired data position. Press the 'Increase' (△) or 'Decrease' (▽) button to set the desired temperature. Once set, press the 'Confirm' button twice to confirm the temperature.

(7) Setting time: Press the 'Confirm' button once to switch from temperature setting to time setting; then press the 'Shift' button once, and the corresponding position indicated will flash. Shift according to the desired data position. Press the 'Increase' or 'Decrease' button to set the desired time. Once set, press the 'Confirm' button twice to confirm the time setting. The time uses a countdown. When the temperature inside the sterilizer reaches the set temperature, the timer starts counting down.

(8) Sterilization: At the beginning of heating, open the lower exhaust valve to the vertical position; when steam emerges from the lower exhaust valve and the pressure gauge indicates 100℃, push the lower exhaust valve to the horizontal closed position; a small amount of steam should be allowed to escape, ideally controlling 15% of the total steam flow. The maximum sterilization temperature used is 124℃～126℃, and the pressure in the range of 0.145Mpa～0.165Mpa is within the safety valve setting. If the pressure exceeds this range, the safety valve will automatically release pressure and start counting the required sterilization time. Upon completion of sterilization, the electronic control device will automatically shut off the heating system, accompanied by a buzzing reminder; and the insulation time will switch to END display; at this time, press the power switch on the control panel to OFF; turn off the power, stop heating and wait for it to cool down.

(9) Drying: After sterilization, items need to be dried quickly. Open the steam release valve and the lower exhaust valve to rapidly discharge steam from the sterilizer, allowing residual water vapor on the items to quickly evaporate.

(10) Open the lid and remove the sterilized items. Close the water source, open the lower drain valve, and drain the water and scale from the sterilization chamber, for next use.

2. Precautions

(1) When stacking sterilized items, it is strictly forbidden to block the steam outlet of the safety valve. Space must be left to ensure clear airflow, otherwise the safety valve will not work due to blockage of the outlet, leading to accidents.

(2) When sterilizing liquids, the liquid should be filled into hard, heat-resistant glass bottles, preferably not exceeding 3/4 of the volume. Use cotton gauze plugs for the bottle mouth, and never use unperforated rubber or cork stoppers. Pay special attention: do not release steam immediately after liquid sterilization is complete; remaining steam must only be discharged after the pressure gauge pointer returns to zero.

(3) Do not sterilize items of different types or with different sterilization requirements together, to avoid compromising one for the other and causing losses.

Experiment 2 Plasmid DNA Extraction - Alkaline Lysis Method

I. Experimental Principle

Bacterial plasmids are a type of double-stranded, closed-circular DNA, ranging in size from 1kb to over 200kb. All plasmids are self-replicating genetic components found in the cytoplasm, independent of the cell chromosome. Under normal circumstances, they remain stably in a free state outside the chromosome, but under certain conditions, they can reversibly integrate into the host chromosome, replicating with the chromosome and being passed on to offspring through cell division.

General methods for plasmid DNA isolation include 3 steps: ① Culturing bacteria for massive amplification of plasmid DNA; ② Collecting and lysing bacteria; ③ Isolating and purifying plasmid DNA. There are many methods for preparing plasmid DNA, commonly used ones include alkaline lysis method, boiling method, SDS method, hydroxyapatite chromatography, etc. In practical operation, the choice can be made based on the host strain type, plasmid molecular size, base composition and structure characteristics, and the purpose of the plasmid DNA. This experiment introduces the alkaline lysis method for plasmid DNA extraction.

Alkaline lysis method for plasmid DNA extraction isolates plasmid DNA based on the topological differences between covalently closed circular plasmid DNA and linear chromosomal DNA. In the narrow pH range of 12.0-12.5, linear DNA double helix unwinds and denatures. Although under these conditions, hydrogen bonds in covalently closed circular plasmid DNA may break, the two complementary strands remain tightly associated, intertwined with each other. When pH is restored to neutral by adding pH 4.8 potassium acetate high-salt buffer, because the two complementary strands of covalently closed circular plasmid DNA remain together, renaturation is rapid and accurate. However, the two complementary strands of linear chromosomal DNA are completely separated from each other, so renaturation is not as rapid or accurate; they entangle to form an insoluble network structure, while the renatured plasmid DNA restores its original conformation and remains in a soluble state. Through centrifugation, chromosomal DNA, unstable macromolecular RNA, protein-SDS complexes, etc., are precipitated and removed. Finally, plasmid DNA in the supernatant is purified by phenol-chloroform extraction.

II. Instruments and Reagents

1. Instruments and Consumables: 37℃ constant temperature shaker, refrigerated centrifuge, benchtop centrifuge, micropipette, 50 ml centrifuge tubes, 1.5 ml centrifuge tubes, pipette tips, measuring cylinders of various specifications, inoculation loops, reagent bottles, 100 l or 250 ml Erlenmeyer flasks, glass rods, etc.

2. Reagents and Preparation:

Preparation of LB Medium:

Yeast Extract 5.0 g;

Tryptone 10.0 g;

NaCl 10.0 g;

After weighing sequentially, add 800 ml of deionized water and stir until completely dissolved. Adjust the pH of the medium to 7.0 with 5 mol/L NaOH (approx. 0.2 ml). Then add deionized water to bring the solution to a total volume of 1000 ml, autoclave at 10 lbs for 20 min, and store at 4℃ after cooling.

Solution I (GET Buffer): 25 mmol/L Tris-HCl (pH 8.0), 10 mmol/L EDTA, 50 mmol/L Glucose

Preparation: 1000 ml

1 mol/L Tris-HCl (pH 8.0) 25 ml

0.5 mol/L EDTA (pH 8.0) 20 ml

20% Glucose (1.11 mol/L) 45 ml

dH2O 910 ml

Mix the above solutions and bottle them. Autoclave at 10 lbs for 20 min, and store at 4℃ after cooling.

Solution II (Denaturing Solution): 200 mmol/L NaOH, 1% SDS

Preparation: 500 ml

10% SDS 50 ml

2N NaOH 50 ml

Take the above solutions, bring to a final volume of 500 ml with sterile deionized water, and mix thoroughly. Store at room temperature. This solution is best used within one month; it is preferable to prepare it fresh.

Note: SDS tends to produce foam, do not stir vigorously.

Solution III (Potassium Acetate Solution): 3 mol/L Potassium Acetate (KAc) buffer, pH 5.6. 5 mol/L Glacial Acetic Acid

Preparation: 500 ml

Potassium acetate 147 g

Glacial acetic acid 57.5 ml

Add 300 ml deionized water and stir to dissolve. After potassium acetate is dissolved, add more deionized water to bring the solution to a final volume of 500 ml. Store at 4℃ after autoclaving or high-pressure sterilization at high temperature and high pressure.

10×TE buffer (pH 8.0): 100 mmol/L Tris-HCl, 10 mmol/L EDTA

Preparation: 1000ml

1 mol/L Tris-HCl buffer (pH 8.0) 100ml

500 mmol/L EDTA (pH8.0) 20 ml

Add about 800 ml of deionized water and mix thoroughly. Bring the solution to a final volume of 1 L. Store at 4℃ after autoclaving or high-pressure sterilization at high temperature and high pressure.

Phenol/chloroform/isoamyl alcohol (25 : 24 : 1): Measure 25 ml Tris-HCl (pH 8.0) equilibrated phenol, add 24 ml chloroform and 1 ml isoamyl alcohol, mix thoroughly, then transfer to a brown glass bottle, and store at 4℃.

Other reagents: TE (pH 8.0), isopropanol, chloroform/isoamyl alcohol (24:1), absolute ethanol, 70% ethanol, ampicillin stock solution (50 mg/ml)

III. Operating Procedures

1. Cell Culture and Harvest

(1) Add 5-10 ml LB medium containing ampicillin (AP) to a 100ml Erlenmeyer flask, then inoculate with a single colony, ensure good aeration, and shake overnight at 37℃ 220 rpm (approx. 16h). It can be subcultured once more and shaken at 37℃ 220 rpm for 3-4 h.

(2) Take 1.5 ml of cultured bacterial solution, transfer to a 1.5 ml centrifuge tube, and centrifuge at 12000 rpm for 3 min using a benchtop centrifuge. Discard the supernatant, invert the centrifuge tube to drain the liquid as much as possible.

2. Plasmid DNA Extraction (Alkaline Lysis Method)

(1) Add 100 ul of Solution I to resuspend the bacterial pellet, pipet up and down until uniformly mixed.

(2) Add 200 ul of Solution II, cap tightly, gently invert the centrifuge tube to mix contents. Let stand at room temperature for 5 min (solution becomes clear and viscous).

(3) Add 150 ul of Solution III, gently vortex for 10 sec, place on ice for 10 min (white precipitate appears in solution).

(4) Centrifuge at 12000 rpm for 6 min, transfer the supernatant to another centrifuge tube (discard pellet).

(5) Add an equal volume of phenol/chloroform/isoamyl alcohol, vortex for 1 min, centrifuge at 12000 r/min for 6 min, transfer the supernatant to another centrifuge tube.

(6) Add 1 volume of isopropanol, vortex to mix, let stand for 10 min, centrifuge at 12000 rpm for 6 min. Discard the supernatant, invert the centrifuge tube on absorbent paper to remove residual liquid drops attached to the tube wall.

(7) Add 200 ul of 70% ethanol to wash the precipitate, centrifuge at 12000 rpm for 2 min using a benchtop centrifuge, discard the supernatant, and air dry the precipitate at room temperature (or in an oven at 50℃-60℃).

(8) Add 20μl TE to the precipitate, pipet up and down repeatedly to fully dissolve the plasmid DNA, and store at -20℃.

IV. Common Problems and Possible Reasons

1. Impure extracted plasmid DNA: Insufficient denaturation; too short reaction time in critical steps; insufficient centrifugation time or speed.

2. Extracted plasmid DNA appears smeared: Excessive force or movement during operation; contamination within the operating system.

3. Incomplete separation from chromosomal DNA: Incomplete denaturation process; problems with reagent preparation (components, concentration, or pH).

Experiment 3 Agarose Gel Electrophoresis

I. Experimental Principle

Agarose gel electrophoresis is a common technique for separating and purifying DNA fragments. DNA samples are loaded into sample wells of a porous support medium (agarose gel) containing electrolyte, and subjected to an electric field. Due to the negatively charged phosphate residues on both sides of the DNA double helix backbone, DNA molecules migrate towards the positive electrode in an electric field. Under a certain electric field strength, the migration speed of DNA molecules depends on the molecular sieving effect. DNA fragments with different relative molecular masses migrate at different speeds, thus allowing separation based on DNA molecule size. Gel electrophoresis can not only separate DNA of different molecular masses but also DNA molecules of the same relative molecular mass but different configurations. During electrophoresis, detection can be achieved by running tracer dyes or relative molecular mass standards alongside the samples. Relative molecular mass standards can provide a benchmark for determining DNA fragment sizes. Adding a small amount of ethidium bromide (EB) to the gel allows its molecules to intercalate between DNA bases, forming a complex that fluoresces orange-red under ultraviolet light irradiation at 254-365 nm wavelengths, thus enabling detection of separated DNA.

Generally, agarose gel electrophoresis is suitable for DNA fragments ranging in size from 0.2kb to 50kb. This experiment introduces the preparation of agarose gel and the application methods of agarose gel electrophoresis in DNA fragment separation.

II. Instruments and Reagents

1. Instruments and Consumables:

Horizontal electrophoresis tank, electrophoresis apparatus, gel imaging analysis system, microwave oven, micropipette, transparent tape, spotting plate or parafilm, 100 ml or 250 ml Erlenmeyer flask, measuring cylinder, pipette tips, etc.

2. Reagents and Preparation:

Preparation of 50×TAE buffer: 2 mol/L Tris-acetate, 0.05 mol/L EDTA (pH 8.0)

Prepare 1000 ml

Tris 242 g

Glacial acetic acid 57.1 ml

0.5 mol/L EDTA 100 ml

After adding 600 ml of deionized water and stirring to dissolve, make up the solution to 1 L. Autoclave and store at room temperature.

Preparation of 1×TAE buffer:

Weigh 20 ml of 50×TAE buffer, then add 980 ml of deionized water.

Ethidium Bromide stock solution: 10 mg/ml Ethidium Bromide

Preparation: 100 ml

Weigh 1 g of ethidium bromide, place it in a 100 ml beaker, add 80 ml of deionized water and stir to dissolve. Make up the solution to 100 ml, then transfer to a brown bottle. Store at room temperature.

6×Loading Buffer: 0.25% Bromophenol Blue, 0.25% Xylene Cyanol FF, 30% Glycerol.

Preparation: 10 ml

Bromophenol Blue 25 mg

Xylene Cyanol FF 25 mg

Glycerol 3 ml

Dilute to 10 ml with 6×TAE buffer, aliquot into 1 ml/tube. Store at -20℃.

Other Reagents: DNA sample, DNA Ladder, Agarose,

III. Operating Procedures

1. Prepare 1% agarose gel (70ml for large gel, 50ml for small gel): Weigh 0.7 g (0.5 g) of agarose into an Erlenmeyer flask, add 70 ml (50ml) of 1×TAE, and invert a small beaker over the flask opening. Heat in a microwave oven and boil 3 times until all agarose is melted, shake well, to form 1.0% agarose gel solution.

2. Gel plate preparation: Take the plexiglass inner tray (gel casting tray) from the electrophoresis tank, wash it clean, dry it, and place the gel casting glass plate into it. Use clear tape to seal the edges of the glass plate and the inner tray at both ends to form a mold. Place the inner tray horizontally and position the comb in the fixed position. Carefully pour the agarose gel solution, cooled to about 65℃, onto the inner tray glass plate, allowing the gel solution to spread slowly until a uniform gel layer is formed over the entire glass plate surface. Let it stand at room temperature until the gel is completely solidified, gently pull the comb vertically, remove the tape, and place the gel and inner tray into the electrophoresis tank. Add 1×TAE electrophoresis buffer until it covers the gel plate.

3. Loading: Mix DNA samples and loading buffer on a spotting plate or parafilm. The final dilution factor of the loading buffer should be no less than 1X. Use a 10 ul micropipette to add samples into the sample wells of the gel plate, replacing the pipette tip after each sample to prevent contamination. Avoid damaging the gel surface around the sample wells when loading. (Note: Record the loading order before loading).

4. Electrophoresis: After loading, immediately apply power to the gel plate for electrophoresis. The voltage should be 60-100V, and samples will migrate from the negative pole (black) to the positive pole (red). As the voltage increases, the effective separation range of the agarose gel decreases. Stop electrophoresis when the bromophenol blue has migrated to about 1 cm from the bottom edge of the gel plate.

(5) After electrophoresis, remove the gel, stain with 1×TAE solution containing 0.5 ug/ml ethidium bromide for about 20 min, then rinse with clear water for 10 min.

(6) Observation and photography: Observe under UV light. If DNA is present, red fluorescent bands will be displayed. Take and save photos using a gel imaging system.

IV. Common Problems and Precautions

1. When preparing agarose, ensure it is completely melted before casting the gel.

2. Agarose gel is fragile, so handle it gently during operation.

3. Pay attention to power lines during electrophoresis to prevent electric shock.

4. Ethidium bromide is carcinogenic. Wear latex or disposable plastic gloves during preparation and use. Use it in a specialized laboratory.

5. UV light is harmful to the human body. Do not keep the lamp on for too long and pay attention to protection.

6. DNA band shape is fuzzy: Too much DNA loaded; voltage too high; air bubbles in the gel.

7. Plasmid DNA exists in three forms: ① Covalently closed circular DNA (cccDNA), often existing in supercoiled form; ② Open circular DNA (ocDNA), where one or more breaks occur in one of the two strands of this plasmid DNA, allowing it to rotate freely to relieve tension, forming a relaxed circular molecule; ③ Linear DNA, caused by breaks in both strands of plasmid DNA at the same site. Therefore, three bands may appear in the results of plasmid DNA electrophoresis, and their migration speeds are: cccDNA > Linear DNA > ocDNA.

Experiment 4 Restriction Endonuclease Digestion and Identification

I. Experimental Principle

Restriction endonucleases are a class of DNA hydrolases that can recognize specific nucleotide sequences in double-stranded DNA molecules, mainly found in prokaryotes. According to their recognition and cleavage characteristics, catalytic conditions, and whether they have modifying enzyme activity, restriction enzymes can be divided into three major types: Type I, Type II, and Type III. Among them, Type II enzymes are the most useful in molecular cloning and gene manipulation and are commonly used molecular biology tool enzymes.

The recognition sequence length of restriction endonucleases is generally 4-8 specific nucleotide pairs that are palindromic. Generally, the longer the recognition sequence, the lower the frequency of recognition sites appearing in the same DNA molecule. The cleavage sites for many restriction endonucleases have been determined. For example, the recognition and cleavage sequence for EcoRI enzyme is the following 6 base pairs.

5′……GAATTC……3′

3′……CTT AAG…… 5′

EcoRI recognizes and cleaves this sequence in a unique way, forming two 5′ overhangs:

and

……G AATTC……

……CTT AA G……

These ends are complementary, i.e., sticky ends, and can be ligated to other molecular ends produced by EcoRI under the catalysis of ligase.

Restriction endonucleases are primarily used for genomic DNA fragmentation, construction and identification of recombinant DNA molecules, isolation and recovery of target gene fragments from vectors, and construction of physical maps of DNA molecules. Depending on the purpose and requirements of digestion, different methods such as single digestion, double digestion, or partial digestion can be employed. Based on the volume of the digestion reaction, it can be divided into small-scale and large-scale digestion reactions. Small-scale digestion reactions are mainly used for plasmid digestion identification, with a volume of 20 μl, containing 0.2-1 μg DNA. Large-scale digestion reactions are used for preparing target gene fragments, with a volume of 50-100 μl and DNA usage of 10-30 μg.

This experiment involves small-scale digestion of plasmid pUC18 with EcoR I. The double-stranded circular DNA molecule of the plasmid has multiple restriction endonuclease recognition sites. After digesting the plasmid with a specific restriction endonuclease, agarose gel electrophoresis is usually used to verify the digestion effect.

II. Instruments and Reagents

1. Instruments:

Water bath, centrifuge tubes, pipettors, tips, electrophoresis equipment, etc.

2. Reagents:

Plasmid pUC18, EcoR I restriction endonuclease, restriction enzyme reaction buffer, agarose, electrophoresis buffer, 6× loading buffer, ethidium bromide stain, sterile water, etc.

III. Operating Procedures

1. Restriction enzyme reactions are generally carried out in sterile 0.2 or 0.5 ml PCR thin-walled centrifuge tubes.

2. Enzyme Digestion Reaction System (10 μl):

Digestion buffer (10×buffer) 1 μl

Restriction endonuclease 0.5 μl EcoR I(7.5U)

Substrate DNA (plasmid) x μl (depends on plasmid concentration)

Sterile ddH2O Add to 10 μl

Add restriction enzyme last, flick to mix or gently pipette up and down with a tip to mix, then centrifuge at 6000 r/min for 15s. Incubate in a 37℃ water bath for 2h.

3. After digestion, take 5 μl of the digestion product for agarose gel electrophoresis to verify the digestion reaction effect. DNA molecular weight markers must be run simultaneously to determine the size of the DNA fragments. If the digestion is incomplete, the digestion reaction can be continued, or an appropriate amount of enzyme can be added and the reaction continued.

5. After observing complete digestion by electrophoresis, place the reaction mixture in a 65℃ water bath for 10-15 min to stop the enzyme reaction. Store the remaining digestion product at -20℃ for future use.

IV. Precautions

1. Restriction endonucleases should be stored at -20℃ and kept on ice during operation to avoid prolonged exposure to high temperatures.

2. Restriction endonuclease solutions usually contain 50% glycerol. After adding to the reaction tube, they often settle to the bottom due to their higher density, so they must be thoroughly mixed.

3. When adding samples, insert the tip vertically into the reagent tube, avoiding contact with the tube wall. Change tips after adding each component, and mark the added samples to prevent errors or omissions and avoid contamination.

4. Enzymes (placed on an ice box) should be added last to minimize exposure to room temperature. When adding enzymes, do not insert the tip too deeply.

5. The temperature and time for the enzymatic digestion reaction should be determined according to the enzyme's instructions.

6. Pay attention to the enzyme dosage. The amount of enzyme added should be calculated as 1-3U/μg DNA, and the volume of the enzyme solution should be less than 10% of the total reaction volume to avoid interference from glycerol in the enzyme solution. When the enzyme amount is too high (≥25U/μg DNA), there is a possibility of so-called star activity, i.e., cutting at sites other than the recognition sequence. Furthermore, star activity may occur when the mass fraction of glycerol in the reaction system is greater than 12%, or in the absence of NACL and the presence of M, among other conditions.

V. Related Questions

1. Enzyme Storage

Different restriction enzymes are often stored in broadly similar buffers. These specially formulated buffers allow the enzyme activity to be maintained for a longer time. The functions of the components commonly used in storage buffers are as follows:

Tris-HCl (7.4―7.8): Maintains a stable pH range.

50-100mmol/L NaCl or KCl: Provides a certain ionic strength.

0.1mmol/L EDTA: Chelates magnesium ions that can activate restriction enzymes.

1mmol/L DTT: Protects reducing groups on enzyme molecules.

200-500ug/ml BSA: Bovine serum albumin increases the protein concentration in the solution, preventing enzyme denaturation due to excessively low protein concentration.

50% Glycerol: Prevents the enzyme solution from freezing at -20℃.

1-5 g/L Triton-100: This is a non-ionic surfactant that prevents surface denaturation of protein molecules.

2. Definition of Enzyme Unit

A restriction enzyme unit is usually defined as: 1U of enzyme can completely digest 1 μg of λDNA in approximately 50 μl of a suitable reaction buffer system within 1h. The specific method for determining restriction enzyme units involves adding a series of enzyme amounts (around 1U) to 1 μg of substrate. When electrophoresis shows that the bands of the digested fragments no longer change, it indicates complete reaction, and the minimum enzyme amount for complete digestion is defined as 1U.

3. Restriction Enzyme Reaction Conditions

In restriction enzyme DNA cutting reactions, the enzyme digestion conditions are crucial for experimental success, including reaction temperature, time, and reaction buffer system. In addition to these conditions, the purity and concentration of DNA also affect the digestion effect. Only when the enzyme reaction conditions are correctly chosen can the optimal digestion effect be achieved.

(1) Reaction Temperature

Early restriction enzyme reactions were all carried out at 37℃. Although this method is simple and uniform, it cannot achieve the optimal reaction temperature for each enzyme. To achieve optimal digestion, the required reaction temperature must be determined based on the chosen enzyme.**

(2) Buffer System

The buffer systems for restriction enzyme reactions are broadly similar, all containing the following components: approximately 100mmol/L Tris-HCl, which maintains the pH of the enzyme reaction system at 7.5-8.5; approximately 10mmol/L MgCl2, which provides activation factors for restriction enzymes; 1mmol/L DTT, which protects reducing groups; and approximately 150mmol/L NaCl, which provides appropriate ionic strength. Due to limitations in understanding, early restriction enzyme reaction systems were only categorized into high-salt, medium-salt, and low-salt based on their NaCl content, which also failed to provide optimal reaction conditions for every enzyme. Many manufacturers now provide 4-10 buffer systems, with only minor differences in their essential components, aiming to maximize the effectiveness of each enzyme.

The buffer is usually prepared at 10x concentration and added at a 1/10 ratio when used. Of course, a 5x buffer can also be prepared independently to reduce pipetting errors.

(3) Reaction Volume and Time

The enzyme reaction volume should generally not be less than 20ul. This is because overly small reaction volumes are prone to errors when adding various components, and glycerol content can easily exceed 10%. Incubation at 37℃ can significantly alter the concentration of components in the reaction system due to water evaporation, thus affecting enzyme activity. At the same time, when performing electrophoresis after the reaction is complete, the amount of DNA in the added sample must be sufficient to show a clear band in the electrophoresis.

Conventional restriction enzyme reactions are generally controlled to proceed within 60 minutes, but the incubation time can also be extended to more than ten hours depending on the target and the enzyme used.

(4) DNA Purity and Concentration

In addition to the enzyme reaction conditions mentioned above, the most important factor affecting the restriction enzyme's effectiveness is DNA purity. For example, if the sample contains a large amount of RNA impurities, it will affect the restriction enzyme's effectiveness by reducing the effective concentration of the enzyme; if certain杂蛋白 (miscellaneous proteins) are not completely removed and bind to DNA, it will also affect the restriction enzyme's effectiveness. As for the various influencing factors not completely removed during the extraction process, there are even more, such as trace amounts of chloroform, SDS, EDTA, phenol, EB, ethanol, or sulfate ions in agarose gel.

Another indicator of DNA purity is that it must not contain trace amounts of DNase. If the electrophoresis result after restriction enzyme digestion shows unclear bands or appears as a red smear (technically called Smear), it can be confirmed that the system has been contaminated with DNase.

If the DNA sample contains a large amount of RNA, it can be treated with RNase; if it contains miscellaneous proteins bound to DNA and DNase, both can be extracted with chloroform/isoamyl alcohol; if the sample contains small molecular substances that inhibit enzyme digestion, such as chloroform, SDS, or sulfate, a new buffer system can be replaced through precipitation. Of course, if enzyme digestion fails, all factors other than DNA, such as the cleanliness of sterile water, EP tubes, pipette tips, and the enzyme activity of the restriction endonuclease itself, cannot be ruled out.

In addition to purity, DNA concentration also affects the restriction enzyme's effectiveness. Generally, a DNA mass concentration of 1ug/20ul yields better enzyme digestion, while a mass concentration as high as 1.5ug/20ul may lead to difficulties in enzyme digestion.

(5) Methods for Terminating Restriction Enzyme Reactions

If subsequent operations such as ligation or labeling are required for the digested fragments, the restriction enzyme must first be inactivated. There are three methods for inactivation: The first method is to add EDTA to the reaction buffer system to a final concentration of 10-12.5mmol/L. The principle is to chelate the magnesium ions required for the enzyme reaction. However, this method will affect subsequent reactions that require magnesium ions. Therefore, after adding EDTA, ethanol precipitation is usually used to change the buffer system to remove EDTA, but this will cause DNA loss. The second method is heat inactivation: general enzymes such as EcoRI can be inactivated by incubation at 65℃ for 60min; some other enzymes such as HindIII require incubation at 85℃ for 30min to achieve the goal; an extreme example is TtbIII, which cannot even be inactivated by heating. The characteristic of the heat inactivation method is its simplicity and that no substances are introduced into the system, making it particularly suitable for continuous enzyme reactions; another characteristic of the heat inactivation method is that it does not require changing the system, so it will not cause DNA loss. The third method is inactivation by phenol and chloroform extraction, followed by ethanol precipitation of the DNA fragments. The characteristic of this method is its vigorous treatment conditions, which can completely inactivate the enzyme (unlike the first method where the enzyme protein is not denatured but merely lacks activation factors).