(1) To understand the routine instruments, equipment, and supplies for the experiment.
(2) To master the function and use of the instruments used in this experiment.
Experimental content
I. Routine instruments and equipment in the laboratory
(I) Temperature control system:
1. Refrigerator: According to the needs of drugs, reagents and a variety of biological agents preservation, it must be equipped with refrigerators at different temperature control levels, the most commonly used are: 4 ℃, -20 ℃, -80 ℃ refrigerator.
4℃ is suitable for storing certain solutions, reagents, medicines and so on.
-20℃ is suitable for certain reagents, medicines, enzymes, serums, prepared antibiotics and DNA and protein samples, etc.
-80 ℃ is suitable for certain long-term low-temperature preservation of samples, purified samples, special low-temperature treatment of digestive solution and so on.
0-10℃ cold cabinet is suitable for electrophoresis, chromatography, dialysis and other experiments under low temperature conditions.
2. Liquid nitrogen tank: some experimental materials, some organs and tissues, cell lines, strains and purified samples, etc., require quick-freezing and long-term preservation in ultra-low-temperature environment, there is a need for a liquid nitrogen tank (-196 ° C) has the advantages of economy, labor-saving and better to maintain the biological characteristics of the cells.
3. Incubator: 37 ℃ thermostat for solid culture of bacteria and cell culture.
CO2 incubator for the culture of various cells, can provide a constant amount of CO2 (usually 5%), used to maintain the acidity of the culture solution (pH).
37℃ constant temperature air shaker can be used for the culture of liquid bacteria.
4. Water bath: used to keep warm.
25-100 ℃ water bath shaker can be used for molecular hybridization test, all kinds of biochemical enzyme reaction and other tests of heat preservation.
25-100 ℃ water bath box for routine tests.
5. Oven: mainly used for drying experimental vessels, some need a higher temperature, some need a lower temperature. For RNA aspects of the experimental apparatus, need to dry in the 250 ℃ oven, some plastic appliances can only be in the 42-45 ℃ oven for drying.
(ii) water purification devices: with the rapid development of molecular biology, many experiments on water purity requirements are increasingly high.
1. Distilled water dish: single distilled water is often difficult to meet the experimental requirements. Double-distilled water, triple-distilled water dispensing, many experiments to deionized water.
Multiple distilled water can remove volatile impurities in the water, can not completely remove the dissolved gas impurities in the water (Mn2 +, Cu2 +, Zn2 +, Fe3 +, Mo (Ⅵ)).
2. Ion exchanger: deionized water - water obtained by ion exchange method, called deionized water.
Deionization effect is good, but can not remove non-ionic impurities in the water, which often contain traces of organic matter (resin, etc.).
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3. Ultra-pure water: distilled water, ion exchange water, reverse osmosis pure water as a water supply, magnet-coupled gear pumps act to make water circulation. Used for PCR, PCR amino acid analysis, DNA sequencing, enzyme reaction, tissue and cell culture.
(C) bacteria sterilization equipment: most of the reagents used in molecular biology, but also experimental utensils should be strictly disinfected and sterilized.
1. Steam sterilization pot: for small quantities of items ready to be sterilized. Large quantities of experimental items, reagents, culture media can be used for large-scale disinfection and disinfection at regular intervals
2. Ultraviolet light: 75% ethanol, 0.1% SDS (disinfectant)
Some of the high-pressure, high-temperature disinfection and can be used for ultraviolet light irradiation or ethanol and SDS immersion.
Ultraviolet irradiation is easy to use and very convenient, but the sterilization effect is related to the distance, and produce ozone contamination safety for sterile rooms, ultra-clean table and plastic utensils disinfection.
3. Filter membrane: not resistant to high temperature, high pressure reagents with its sterilization.
4. Boiling sterilization: mainly used for metal instruments and needles when urgently needed.
(D) measurement system:
1. weighing system: (a variety of balances) bench scales, pallet balances, knob swing balance,
photoelectric molecular balance, precision electronic analytical balance
2. liquid volume measurement: fine: measuring cylinder, pipette, microfuge
crude: graduated test tubes, beakers, conical flasks
3. pH measurement:
4. 3. pH measurement:
PH meter: the instrument for determining the direct potential of H+ in solution, mainly through a pair of electrodes, in different pH solutions to produce different electric potentials expressed in pH.
pH test paper: only for culture solution, phenol saturated solution, buffer or other reagents solution of the pH value of the rough estimate. The preparation of most reagents requires a strict pH value, requiring high accuracy (two decimal places) pH meter.
4. OD value measurement: optical density, spectrophotometer is the use of substances in the visible and ultraviolet region of the absorption spectrum to identify the nature of the substance and its content of an instrument. It is composed of a light source, monochromator, absorption cell, receiver, measuring instrument or display screen. OD value is one of the many solutions of solutes in the quantitative convenience index, through the monochromatic light produced and determine the absorption value of a solution to the monochromatic light, using it can be quantified and the purity of the nucleic acid solution of the preliminary judgment.
(E) centrifuge: centrifugal technology is an indispensable means of physical technology in the study of biological structure and function. Because of the various substances in the sedimentation coefficient, buoyancy, and mass and other differences, can use a strong centrifugal force field, so that their separation, purification and concentration. There are a variety of centrifuges available. They are available for centrifuging samples from less than 0.05 ml to several liters. Centrifugal techniques are used for a wide range of applications, including the collection and separation of cells, organelles and biomolecules. According to their different rotational speeds, they can be divided into the following types:
1. Ordinary centrifuges: Maximum rotational speed of 6000 r/m , maximum centrifugal force of 6000g
①Medical or tabletop centrifuges: are the simplest and cheapest of centrifuges, and are most commonly used for the collection of substances with rapid sedimentation coefficients, such as erythrocytes, coarse precipitates, yeasts and bacteria, and so on.
2 ② low-speed freezing centrifuge: mainly used for cells, nuclei, cell membranes and bacteria precipitation and collection, etc.. 2
2. High-speed centrifuge: maximum speed 25000 r/m , maximum centrifugal force 89000g
There are two kinds of freezing and room temperature, mostly used for preparation and hand collection of microorganisms, cellular debris, cells, large organelles, ammonium sulphate precipitates, and immunoprecipitates, etc.
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3. ultracentrifuge: maximum speed 9000 r/m, maximum centrifugal force of 694000g.
4. tabletop ultracentrifuge: maximum speed 12000 r/m, maximum centrifugal force 625000g.
(6) ultra-clean bench: inside the ultraviolet lamp, lighting, there should be a flame of alcohol lamps, 75% ethanol and other sterilizing equipment. Is a kind of equipment to provide local cleanliness. The principle is that the blower drives the air, after low, medium-effect filters, through the worktable, so that the experimental operation area becomes a sterile environment. Ultra-clean table according to the different airflow direction of the roughly several types:
①Lateral flow: purified airflow, from the left or right through the work surface, flow to the opposite side, or from the top to the bottom or from the bottom to the top of the flow to the opposite side, they can form a barrier to protect the surface of the airflow and sterility.
Disadvantage: at the junction of the purified gas flow and the outside gas, there can be a negative pressure due to the flow of gas, so that a small amount of unpurified gas is mixed in, resulting in contamination.
②Outflow type: the gas flow is facing the direction of the operator to ensure that the outside gas can not be mixed.
Disadvantage: when conducting experiments on harmful substances, it is unfavorable to the operator, but organic glass can be used to cover up the upper part, so that the airflow flows out to the bottom. (VII) electrophoresis system: electrophoresis technology is the detection, identification of a variety of biological macromolecules, purity, content and description of their characteristics, and even or separation, purification, recycling and concentration of samples is one of the tools. Nucleic acids and proteins, among others, carry an electric charge and are able to move when they are placed in an electric field.
The electrophoresis unit consists of two parts: a power supply unit and an electrophoresis tank unit.
① Electrophoresis device: the power supply needs to be stabilized through a voltage regulator, which can provide both a stable direct current and a stable voltage output. It can be used for three kinds:
Normal degree stabilized electrophoresis device: output voltage 0-500v 0-15mA
Medium degree stabilized electrophoresis device: output voltage 400-1000v
High degree stabilized electrophoresis device: power supply device with output voltage more than 1000.
② Electrophoresis tank device: there are two kinds
Horizontal electrophoresis tank: generally divided into miniature electrophoresis tanks and large horizontal electrophoresis tanks
Vertical electrophoresis tanks: divided into vertical flat electrophoresis tanks and cylindrical electrophoresis tank device.
(H) PCR instrument: Polymerase Chain Reaction instrument, also known as DNA thermal cycler, gene amplification, it is to make a pair of oligonucleotide primer binding to the positive and negative DNA chain on both sides of the target sequence, thus enzymatically synthesized copies of millions of times the target sequence of the DNA fragments, it is a cycle of each cycle, including DNA denaturation, primer denaturation, DNA polymerization, DNA polymerization, DNA denaturation, DNA polymerization, DNA polymerization, and so on. Each cycle consists of three processes: DNA denaturation at three different temperatures, primer reversion, and DNA polymerase-catalyzed elongation reactions.
(ix) Gel Imaging System:
Observation and analysis of ethidium bromide (EB)-containing nucleic acid samples after electrophoresis.
(x) Drying equipment:
1. Vacuum heating drying box: nucleic acids are fixed on nitrocellulose membrane and nylon membrane for hybridization experiments.
2. electrophoresis gel drying box: dehydration and drying of the gel after electrophoresis apparatus, generally can be dried to some cellophane, dried gel is easy to save.
3. Liquid nitrogen freeze drying: suitable for drying and crystallization of active protein samples.
4. Vacuum pump: many experiments require vacuum. For example: drying of nucleic acid samples after ethanol precipitation, drying of electrophoresis gels, etc.
(XI) Other
1. microwave oven: easy to facilitate the rapid heating and heating of some solutions, electrophoresis agarose gel preparation, dissolution and so on.
2. Ice machine: used to create a low-temperature environment required for most experimental operations on nucleic acids and proteins to reduce the hydrolysis of nucleases or proteases.
3. Chromatography device: (chromatographic separation) is an effective physical method for separating multi-component mixtures.
Vacuum imprinting system, DNA synthesizer/sequencer: these are essential instruments for in-depth study of nucleic acids.
4. Magnetic Stirrer: Multi-Angle Rotary Mixer, Rapid Oscillatory Mixer: these are used for mixing instruments.
5. Tissue homogenizer: ultrasonic tissue and cell crusher, with which the sample separation and purification experiments.
6. Ventilation cabinets: many solvents can escape toxic gases, the necessary cabinet, radioactivity tests should also have Plexiglas shielding.
7. Glass stills, electric heating plus cap, transformer: used for distillation of organic solvents such as phenol.
8. Tip head, Eppendorf tube:
Microtube pipette tip head (pipette tip), Eppendorf tube (microcentrifuge tubes) can be washed, silicone disinfection after repeated use. For some demanding experiments, such as RNA extraction, preservation and other operations, new sterilized tip heads with Eppendorf tubes should be used. In addition, there should be commonly used specifications of centrifuge tubes (1000 ml, 500 ml, 250 ml, 50 ml, 7 ml, etc.) and 96-well, 24-well, 12-well, 6-well plastic plates for cell culture.
9. Small equipment, utensils:
Timer, filter film, plastic wrap, protective glasses duckbill tweezers, conventional glass or plastic utensils (including flatware, test tubes, beakers, measuring flasks, reagent dispensing funnels, light-protected reagents should be used to save the brown reagent bottles, such as saturated phenol, mercaptoethanol, etc.), marking pens, a variety of gloves PE, latex, household, acid-resistant, etc.)
< p>Second, the main instruments used in this period of experimentation, the function and operation of equipment
1. Phenol redistillation device: air condensing glass still.
General phenol is a colorless transparent crystals, such as pink or yellow, indicating that it contains phenol oxidation products, such as quinone, diacid, etc., discoloration of phenol can not be used for experiments, because of which phenol oxides can destroy the phosphodiester bonds of nucleic acids, and cause cross-linking of the DNA chain. Before use, it must be redistilled at 160℃-180℃ to remove the contaminating impurities that can cause RNA and DNA breakage and cross-linking, so as to obtain pure phenol.
2. Magnetic stirrer: 81-2 type constant temperature magnetic stirrer
Connect the conductive temperature meter with a wire and a two-core plug, insert it into the temperature meter hole at the back, and then it will stop heating automatically when the temperature rises to the pre-adjusted degree (temperature-controlled stirring). Mainly used for rapid stirring, dissolving and mixing of substances. Such as the preparation of reagents, the preparation of water-saturated solution of phenol. 4
3. water distiller: 1810B automatic double pure water distiller, quartz tube heating type, the device of the primary and secondary distillation are protected, safe and reliable, thermal relay when the cooling water suddenly interrupted or lack of water, that is, automatically cut off the power supply. Reed relay controls the water level in the secondary distillation bottle, the height of which should be able to automatically cut off the power supply when the water level is lowered, so as to achieve the purpose of automatically controlling the water level.
The instrument is mainly used for the preparation of double and triple distilled water.
4. Centrifuge: TGL-16G desktop high-speed centrifuge, maximum speed of 20,000rpm, mainly used for the precipitation of proteins and the layering of the organic phase and aqueous phase in nucleic acid extraction, instantaneous centrifugation for PCR detection.
5. Microfuge: Mainly used for precise measurement of micro liquid volume and transfer of aqueous phase of nucleic acid.
6. homogenizer: 5ml, 10ml glass homogenizer.
Mainly used for tissue cell crushing.
7. Gel imaging analysis system
Mainly used for nucleic acid samples agarose gel and PCR electrophoresis results of UV observation and illumination.
8. PCR instrument: PTC-200 gene amplifier
In vitro amplification of nucleic acids
9. Steam sterilizer: used for experimental reagents, utensils, appliances and other sterilization and disinfection
There are two types of electric heating, electric furnace heating.
10. Ultra-clean table: JJT-1300 clean bench, side-flow type.
Principle: suction inlet air through the primary and secondary filter dish (non-woven filtered), and then through the fan by the senior filter (made of ultra-fine glass fiber filter paper) and the formation of clean air. Mainly in the local air to create a clean air environment, in order to work in the area of suspended particles and microorganisms are controlled to a minimum.
The operating pressure of this table is vertical laminar pressure, so the air outlet surface and the operating position should not be placed in excess of the items, so as not to impede the normal flow of airflow, affecting the degree of cleanliness, the holes in the table panel as a ventilation holes, the use of liquids or other items to avoid leakage into the air, so as not to affect the internal components. The wind speed is adjustable.
Use: 75% ethanol disinfection 2-3 times, arrange the experimental supplies. Plug in the power supply, UV disinfection for 30 minutes, start the fan 5m and turn off the light, turn on the lighting 10 minutes after turning off the light, you can use it.
11. Electrophoresis system:
①Electrophoresis instrument: HV-3000 type height three constant multi-purpose electrophoresis instrument (constant pressure, constant current, constant power)
②Electrophoresis tanks: horizontal two.
12. Microwave oven: mainly used for rapid dissolution of agarose.