中洪博元,动物造模 z-bo 2020-07-21 837

　　Many friends who cooperate with Zhonghong Boyuan in animal modeling will ask, how do you prepare reagents when modeling?

      First, let us divide the types of reagents that need to be used in animal experiments according to storage conditions. Some reagents can be used, while others require preparation.

　　The former, etc.: more stable or single-dose packaged reagents. Western DAB color reagents are easily available and can be used directly. The latter, for example: most drugs and reagents. Of course, most of them require preparation.

　　 is classified according to its use, and can be classified as follows. Drugs: Compounds or Chinese herbal extracts that usually study the efficacy or mechanism of compounds. It can be divided into raw materials and preparation. The API is usually directly available. Some preparations need to be extracted, and some can be used directly.

Animal model reagents: STZ is used for diabetes, galactosamine, carbon tetrachloride damage to the liver, cadmium damage to the testis, bleomycin damage to the lungs, adenine damage to the kidneys, and carrageenan Inflammation

　　 Animal model feed: usually this is solid. Some are semi-solid. The liquid state is low. For example, a high-fat diet should be mixed with cholesterol, propylthiouracil, cholate, egg yolk, lard, etc.

Basic animal manipulation reagents: such as anesthetics, animal depilatory agents, sodium sulfide, animal markers***, heparin reagent for blood collection or intubation, 75% ethanol for disinfection, iodophor, promethazine, animal surgery antibiotic

Various buffers or basic dispersion media: such as PBS, saline, citrate buffer, Krishna solution, Ren solution, 0.5% CMC-Na solution, DMSO solution, adding solvent poloxamer (mixed with drugs, Then added to the solvent)

　　Reagents used to store specimens: such as 10% formalin, neutral formalin, 4% paraformaldehyde, Brinell solution, etc.

　　Cleaning solution: such as dilute hydrochloric acid solution, potassium dichromate lotion.

　　 Detection reagents: vary with experiment. Each experiment has a different method. You can open another special discussion.

　　 is divided according to its usage time limit.

　　 is a ready-to-use type. For example: STZ is very easy to disassemble and should be used up immediately after preparation. Otherwise, it will be completely disabled. Some enzyme reagents should be used up as soon as possible after preparation. Well-prepared developers usually run out in a short period of time. Drugs unstable to water, such as penicillin. Please use it up as soon as possible.

　　You can use the short film within a week. Mainly some metastable solutions. It will slowly decompose under the action of temperature and oxygen, and cannot meet the requirements of use. For example, ammonium persulfate in protein electrophoresis is an auxiliary reagent that can promote the formation of oxygen free radicals and promote the formation of gels. Free radicals are not generated for a long time, so it is usually used for 1 to 2 weeks. Under cryopreservation, some buffers will be exhausted as quickly as possible, especially in in vitro experiments. Otherwise, it may affect the experimental results.

　　 Medium efficiency type within 1 month: If stored at low temperature after preparation, many reagents can be used for 1 month or longer.

　　Long-acting for more than 3 months: Many solutions with stable performance and low concentration accuracy requirements can be stored for a long time. Brine and CMC-Na solution and formalin.

　　 can be classified as follows according to its purity.

　　Chemical purity: used in general chemical experiments with a small amount of impurities. The purity should generally exceed 98%. Analytical purity: In the analysis experiment of trace impurities, the purity exceeds 99%. Abbreviation: AR

　　 Chromatographic purity: requirements for liquid mobile phase. As far as I know, there should be no impurity peaks. The purity can be 99.9%.

　　 Biochemical purity: It seems to be written on some biochemical reagents. Abbreviation: BR

　　 Please note that some manufacturers mark the GR grade. In fact, this can be equivalent to analytical purity.

　　 is the highest level: standard products, reference products. In fact, there is not much difference between the two. Usually, these two words are used together. According to the Pharmacopoeia, the reference materials we usually use are called reference materials. Antibiotic reference substances (note that they are not synthetic antibacterial agents) are called standard substances. In fact, we always seem to regard “standard products” as the highest level, and “reference products” as lower than “standard products”. This is not the case, the two words must be the same. Only standard products specifically mention antibiotics or other biological products. Which resulted in

　　Distinguish the two:

　　Reference materials, reference materials refer to reference materials used for identification, inspection and content determination. Among them, the standard substance refers to the reference substance used to measure the content and potency of bioassays, antibiotics and biochemical reagents, calculated in titer units and calibrated with international standards. For example, the reference substance of penicillin is called the standard substance, and the reference substance of ofloxacin is called the reference substance. However, in the published papers, most of the situations we see are a mixture of the two. Some people use penicillin as a reference and standard.

　　 Then what are the requirements for these reference materials? I think the purity must be 99.99% or higher. This is not true for all biological agents or antibiotics, because some antibiotics are actually mixtures. Only through efficacy evaluation.

　　 When preparing standard products or reference products, please pay special attention to accuracy. When weighing less than 10 mg of material, it is often difficult to be accurate. Therefore, when preparing reagents, the accuracy can be improved by increasing the concentration of 10 mg or more by weight and gradually diluting.

　　 Each reagent has different regulations and requirements for different contents. You will see on the paper

　　Reagent purity. Usually write articles about chemical purity, analytical purity, chromatographic purity and purity. How many marks are included? This requires understanding the purity requirements of the reagents used before conducting the experiment. Otherwise, it is difficult to obtain satisfactory results. For example, if chromatography requires you to use pure reagents and analytical reagents, the results of the experiment may fail.

　　Analytical level or above used in animal experiments. When conducting pharmacokinetic experiments, please pay attention to liquid phase detection and use chromatography reagents. Otherwise it will seriously affect your experiment results. According to toxicity, there are several types of non-toxic, low-toxic, addictive and high-toxic.

　　 Please store and protect toxic reagents carefully during the experiment.

　　 Next, let's talk about the storage of reagent materials.

　　The storage method mentioned here is the storage method until the reagent provided by the supplier is used. In the past, many people did not pay much attention to this issue. But this is an important question. Otherwise, the experiment has not started yet and the reagent has failed and should be replaced temporarily. Or, during the experiment, the reagent may suddenly become unavailable, and the entire experiment may fail. Therefore, it is very necessary to carry out this preliminary work.

　　 According to the state, there are solid, liquid and semi-solid. Solids are relatively easy to store. Special attention should be paid to liquids and semi-solids.

　　 Most reagents are solid. Some solids are crystals, and crystals appear in many forms, including needles, columns, and small crystals. Some are granular, some are bumpy. Among them, acicular crystals and columnar crystals have good fluidity, and sheet and block shapes are not very good. In the future, the weight will have a certain influence when preparing reagents. The storage of this type of material is sealed. If necessary, please store strictly.

　　 There is a solid type that easily absorbs water. Like calcium chloride or zinc chloride. Pay special attention to the influence of relative humidity.

　　 There are several types according to its storage requirements: room temperature storage, low temperature freezing, normal freezing, low temperature freezing and deep freezing (less than -70 degrees).

　　Reagent materials stored at room temperature can be placed in the cabinet where the reagents are stored in the laboratory. Generally, avoid light and avoid overheating. Generally, please be careful above 35 degrees. In addition, it should be noted that different grades of reagents need to be separated. .. Please separate those that are easy to absorb and those that are difficult to absorb. Place it in an obvious and easily accessible location. Under certain conditions, hazardous and toxic reagents that may explode should be stored separately.

　　 There are two things to note about freezing at low temperatures. First of all, do not freeze frozen food. Generally speaking, refrigeration will not freeze because the temperature is usually between 0 and 4 degrees. Of course, this refers to reagent materials that partially contain water. The second problem is that the raw materials of common refrigeration reagents are too stable, but they cannot be frozen because they will lose or reduce the activity of certain types of substances, so they should usually be used as soon as possible.

　　 Normal freezing usually means placed at 0 degrees. Low temperature freezing usually refers to storage at -20 degrees. These two storage methods are usually suitable for certain enzymes that are unstable at room temperature. Do not close the refrigerator loosely or open the refrigerator repeatedly in a short time. The reagent material may be inactivated. In addition, before deicing the headbox, it is necessary to move it to the corresponding temporary storage under the same conditions.

　　Cryogenic storage is usually suitable for materials that need to remain active for a long time. For reagent raw materials, it is usually rare. This is because the solid content of the reagent raw materials occupies the most. The general decomposition of solids is relatively slow. It is more often used when storing experimental specimens. According to toxicity, there are several types of non-toxic, low-toxic, addictive and high-toxic. Therefore, it is necessary to pay attention to protection during use and storage.

　　 For toxic raw materials, the first thing to do is to understand and understand its toxicity mechanism. Some can be frustrating, such as redness, burning and other adverse reactions. Some can cause blood toxicity, for example benzene can affect granulocytes. Some are neurotoxic, such as acrylamide monomers. Some are carcinogenic, which is our greatest concern. Example: Ethidium bromide EB, PMSF and benzyl sulfonyl fluoride are commonly used protease inhibitors, and the commonly used anesthetic uranium oxygen also has specific carcinogenic activity. Second, we need to know how to protect. Be careful when handling and preparing. You can usually wear disposable gloves. Third, unfortunately, you need to know what to do if you are infected with these ingredients. As long as they can be found early instead of in the mouth, basically all substances can be processed and washed by themselves. Example: Wash with a lot of water, choose the correct detergent, and use special effect substances for attenuation treatment. Fourth, we must know how to deal with these substances after use. Don't cause serious pollution to society.

　　 Above, we described the process of storing reagent materials as needed. Next, we will discuss the general feel of packaging reagent raw materials.

　　How do I know if there is a problem when I have reagents? Do you want to indicate whether the packaging is damaged? Is it a regular manufacturer? Is it overseas import or domestic subcontracting? Does the shape meet our requirements? Are there any special requirements during transportation? Does the specification meet the requirements? Generally, there are four methods for obtaining reagent materials. One is to go to the reagent supply point to buy or receive it. For some commonly used reagents, some schools or research institutions have reagent warehouses that can be collected after completing the relevant steps. Some can be purchased at a reagent store. These can directly see the packaging logo. Second, the reagent sales staff will deliver it to the door. Basically, you can run it normally. The third is mail order. Most of them are sent by express. Please check after inspection. If you have any questions, you can contact your supplier again. The fourth is a gift from others. Normally, we will take this into consideration and recommend that you check the label on the original packaging. Please note that some biochemical reagents in particular have expired.

　　 Please refer to the packaging of reagent materials. Varies by manufacturer, batch number, specification, and quantity. The effects of reagents from different manufacturers may vary. Different batch numbers may also be different. Specifications are also important. tailor. Don't waste too much reagents. However, multiple sampling will result in a certain loss, which is higher than the actual loss. Sometimes the courier misses things and misses them, which is also troublesome. For multi-layered reagents, remember that a smaller package is sufficient.

　　 The above are suggestions for reagent materials, but please carefully consider these suggestions at the beginning of the experiment, and use the materials strictly according to your requirements. Third, the solubility of the reagent.

　　 This applies to most solid reagents. When it is a liquid reagent, it depends on whether it can be mixed or dispersed in a solvent.

　　 Generally speaking, reagents can be divided into two categories: soluble and insoluble. It can be divided into two categories: lipophilic and hydrophilic.

　　 Let's talk about how to prepare these reagents.

　　Easily soluble: Like most acid, alkali and salt reagents, it is easily soluble and usually water-soluble. Many of these reagents are prepared in double distilled water.

　　 Insoluble: Some insoluble substances are highly fat-soluble and can be mixed with oil. However, sometimes oil cannot be used, but at this time it must be mixed, so a suspending agent or solubilizer must be added or a co-solvent must be used. Suspending agent, for example: carboxymethyl cellulose sodium (CMC-Na); solubilizer, for example: Poloxamer 188; co-solvent, for example: ethanol. Some have DMSO. DMSO is called a universal solvent. However, please note that in actual use, it may cause pharmacological effects and may interfere with experimental results. If possible, do not use.

　　In the preparation of insoluble matter, it can be heated, stirred or sonicated. Please pay special attention not to blindly heat. One is a substance that rapidly degrades to produce decomposition products when heated, and the other is a substance that becomes ineffective when heated, such as enzymes or protein-like substances. The solubility of certain substances is well known. Of course, you will be familiar with this kind of content. However, some reagents are unknown. There are three ways to do it. One is to check the instructions on the reagent package. Usually, you will find some of the above information. The second is online inspection. Web pages or papers found on Google. The third is to try. You can try a small amount. Look at the solubility. I used to use testosterone propionate injection and wanted to dilute it, so I diluted it with double distilled water. It turns out that it is not mixed at all. At the time, this was very clear and incredible, because the instructions did not specify the prescribed solvent. After that, it is diluted with vegetable oil and the effect is good.

　　Sometimes, I think I know more about the pharmacology of chemistry and the solubility law from a structural point of view. However, due to overconfidence, some exceptions are usually ignored. Like berberine hydrochloride, it must be structurally water-soluble. However, it is actually insoluble. Think about it, ikarin glycosides have glycosyl groups. Glycosyl is very polar and always soluble, right? However, it is almost insoluble in water, but soluble in ethanol and methanol. Therefore, in addition to referring to relevant information, the solubility of the substance is indeed very important, and some preliminary tests must be prepared. It is worth noting that

　　Ethanol is a very good solvent. It is usually mixed with various concentrations of water, but in some cases it may be more satisfactory.

　　 Insoluble: Some insoluble substances are highly lipophilic and can be mixed with oil. However, sometimes oil cannot be used, but at this time it must be mixed, and a suspending agent or solubilizer must be added or a co-solvent must be used. Suspending agent, for example: carboxymethyl cellulose sodium (CMC-Na), solubilizer, for example: Poloxamer 188, cosolvent, for example: ethanol. Some have DMSO. DMSO is called a universal solvent. However, in actual use, please note that it may interfere with pharmacological effects or experimental results. If possible, do not use. \r\nWhen preparing insoluble matter, it can be heated, stirred or sonicated. Take special care not to overheat. One is a substance that quickly decomposes to produce decomposition products when heated, and the other is a substance that loses its function when heated, such as enzymes and protein substances. The solubility of certain substances is well known. Of course, you will be familiar with this kind of content. However, some reagents are unknown. There are three ways to do this. One is to check the instructions on the reagent package. Usually, you will find some of the above information. The second is online inspection. Web pages or papers found on Google. The third is to try. You can try a small amount. Look at the solubility. I have used testosterone propionate injection before, so I wanted to dilute it, so I diluted it with double distilled water. You will see that they are not mixed at all. This is very clear and unbelievable, because the stated solvent was not specified at the time. After that, it is diluted with vegetable oil and the effect is good. \r\nSometimes, I think I know more about chemical pharmacology and solubility laws from a structural point of view. However, due to overconfidence, certain exceptions are usually ignored. Like berberine hydrochloride, it must be structurally water-soluble. However, it is actually insoluble. please think about it. Icarin glycoside has a sugar group. The glycosyl is very polar and will always dissolve, right? However, it is almost insoluble in water, but soluble in ethanol and methanol. Therefore, in addition to referring to relevant information, the solubility of the substance is of course very important, and some preliminary tests need to be prepared. \r\nEthanol is a very good solvent. It is usually mixed with different concentrations of water, but in some cases it may be sufficient. \r\n? Fourth, let’s talk about the solvents or dispersion media commonly used to prepare reagents.

　　It must be said that this is a problem that everyone knows.

　　The only commonly used solvents or dispersion media are water, organic solvents, complex solvents and buffers.

　　Water is the most widely used solvent. Of course, tap water (at least distilled water) cannot be used to prepare reagents. It is best to use double distilled water. Double distilled water is actually equivalent to water for injection. Double distilled water can meet the requirements of sterility and is pyrogen-free. When preparing water-soluble reagents, care should be taken to clean and disinfect the relevant containers. Please note that you cannot measure at high temperatures. Deformation will cause inaccurate measurement results. Etc.: measuring cylinder, measuring cup, measuring bottle. Usually this kind of glass container is immersed in a dichromic acid solution and then washed.

　　Organic solvents are essential for many reagents. Mainly use ethanol, methanol, acetone and chloroform. Except for the occasional use of ethanol, the rest is rarely used as a solvent for oral drugs in animals. Use pure analysis. Of course, you must use chromatographic purity for liquid phase detection. Sometimes, these solvents can be used as the reagent itself without any preparation. Etc.: Precipitate proteins with methanol. When performing PCR, the protein is removed with chloroform. Please wash with ethanol. Pay attention to the flammability and toxicity of organic solvents. One is to prevent fires caused by improper operation, and the other is to take certain protective measures, such as wearing a mask or disposable gloves. Pay attention to the toxicity of methanol to the eyes and the liver toxicity of chloroform. These toxicities are far lower than carcinogens, but they cannot be ignored. The compound solvent usually refers to a highly hydrophilic solvent, such as a mixture of ethanol and water. Acetone can also be used, but use it with caution. Certain reagents can be prepared better with specific concentrations of ethanol. If you want to measure the concentration of ethanol more accurately, it is best to use an alcohol hydrometer.

　　 Strictly speaking, the buffer is a dispersion medium, not a solvent. However, it is widely used in pharmacological experiments. This is because certain reagents must be stable under certain pH conditions. The most commonly used solution is phosphate buffered saline (PBS). PBS can also adapt to different pH values. Also, acetic acid-sodium acetate buffer and citrate buffer. The aforementioned STZ must be stable between pH 4 and 4.5 and must be buffered. At the same time, some buffers are a solution system for washing or temporarily storing tissues. For example, when collecting tissue samples, normal saline or PBS is usually used to clean blood. Similarly, like running buffer, it is also the basic medium for protein electrophoresis and DNA electrophoresis. Please pay attention to the following points when preparing the buffer zone: 1. Please strictly follow the instructions. The order of the substances mainly added to the solution. Due to the wrong order, it may be unusable due to precipitation and other phenomena. The second is to accurately measure the PH value. The third is to build strong labels. When preparing the protein running buffer, invert the concentrated gel buffer and separation gel buffer once, which may cause errors. The two have different pH values due to their different component ratios. Bright and powerful label, easy to identify.

　　 Fifth, let's talk about the selection of weighing, measuring and other reagent preparation equipment. As we all know, most of the raw materials of reagents are solid, while some are liquid. The preparation of solids requires weighing and measuring equipment, while the preparation of liquid materials requires measuring equipment, some of which require weighing equipment. Glass rod, magnetic stirrer, PH test paper, separatory funnel, filter paper and other equipment should also be used in the preparation process to adapt the reagents to your requirements.

　　 Let us talk about this issue.

　　Use weighing equipment

　　 Most reagents are solid. When mixing solids into reagents, metering equipment is usually used. The weighing equipment is usually a balance. There are narrow and relatively accurate steel mills, but they are used in pharmacies.

　　 balances have a variety of working methods, including conventional tray balances, torque tray balances, photoelectric mechanical balances and electronic balances. Ordinary balances are usually used for rough weighing, and everyone is used to it. Torque balances are also easy to use, while photoelectric analytical balances are rarely used. The following focuses on electronic balances.

　　The balance has multiple precisions, including 1g, 0.1g, 1/100g, 1/1000g, 1/10000g and 1/100000g. In this way, we must pay attention to what requirements the weighed raw materials must meet and choose the appropriate accuracy. It is obviously inappropriate to choose an accuracy of 0.1g to 0.01g. Choosing 1/1000 is acceptable, but using 1/10000 is unnecessary and inappropriate.

　　 Balance range. Each balance has a range. It is not appropriate to exceed or approach the maximum range. Of course, your weight cannot fall below the minimum range. Confirm before use.

　　Balance adjustment level. Adjusting the level is a task that must be completed before using the balance. The main premise is that the room where the balance is located must control the temperature and humidity, and the table where the balance is located must be level. Please read the instructions carefully and make adjustments. Reading and adjusting the manual can save a lot of time. The level gauge should be located directly behind the balance, and the line of sight should be perpendicular to the balance. Please pay attention to the blisters of the central level gauge. Make adjustments carefully and patiently. Don't be too rude about using

　　 Description of the balance. In addition to the above understanding of accuracy, range and adjustment level, there are always some precautions to keep in mind during the weighing process. One is to plug in the power for a few minutes in advance, and then wait until the display on the electronic balance is stable before weighing. Secondly, except in some special cases, reagent materials are usually added laterally. Third, weigh aid. Weighing paper can usually be used to weigh solids or containers; containers can be used to weigh liquids. Fourth, weigh and clean up the remaining solids and accidental spilled liquid. Brush off solids. You can use absorbent paper to dry and absorb the liquid, replace the paper and wipe gently.

　　 For solids, usually use a small spoon to grind them into powder or small particles, and then use a corner spoon to gently shake on the weighing paper or container. When weighing highly hygroscopic solids, pay special attention to the humidity of the environment and weigh quickly. For some liquids that need to be weighed, use a dropper or pipette to add. Some solids are crystals, and crystals appear in many forms, including needles, columns, and small crystals. Some are granular, some are bumpy. Among them, acicular crystals and columnar crystals have good fluidity and are easy to pour and weigh. The scale and pellets should be weighed carefully. Normally, the poured reagent will not be poured into the original bottle. Additional installation may be difficult to guarantee the shelf life. Therefore, please pay attention to avoid waste when weighing raw materials.

　　 Use of B measuring instrument

　　 Main measuring instruments: graduated beaker, graduated test tube, graduated pipette, graduated cup, graduated cylinder, graduated bottle, pipette, pipette*, micro sampler.

　　 The first four are relatively rough scales, which are basically estimates. Reagents that do not require precise concentrations can be formulated.

　　The specifications of the graduated cylinder are different. Please choose according to your situation. It can be used to prepare solutions of any volume within the specified range and is the most common method for preparing solutions.

　　 Volumetric flask is more accurate. But it cannot match any capacity. It has only some quantitative size specifications. Etc.: 10ml, 25ml, 50ml, 100ml, etc. are mainly used for the preparation of standard solutions. There are colorless and brown glasses.

　　 Pipette is more accurate. There are many specifications. When using, please use ear wash bulb to absorb, adjust the volume with your fingers and put it down. This requires some adaptive training. Practice makes perfect. Due to the widespread use of pipettes* today, it is necessary to understand the advantages of pipettes in certain aspects. Proficiency can increase the efficiency of your experiment.

　　Pipetting* is a measuring instrument that has been widely used in laboratories in recent years. Especially in the absorption of certain trace solutions, it plays an irreplaceable role in certain experiments where pollution must be strictly avoided. When using a pipette*, please be careful to match the *head and *, and be careful not to suck in the liquid* when aspirating. *Be careful when adjusting the dial. When not in use, please adjust to the maximum range. It must be calibrated regularly during use. There are imported products and domestic products. Imported quality is better.

　　 Micro sampler is also a precision measuring instrument. It is mainly used for liquid phase injection and protein electrophoresis injection. It has the function of inserting the device during use or when the liquid level is below a certain level. There are 20ul, 50ul and 100ul ranges. Be careful not to bend the long metal mouth forward or block insoluble materials. Also, do not pull out the piston rod. After use, wash with ethanol or distilled water and put in a special box.

　　 The glass measuring device cannot be fired at high temperature to avoid inaccurate capacity. Blow dry only. Pipetting* and micro syringes should be maintained according to instructions.

　　C Use other tools

　　Using a beaker Generally, a beaker is used when preparing reagents. Especially for reagents that require heating, some solvent (usually water) is usually added to dissolve everything, and then a solvent is added to make up. Pay attention to the size of the beaker and put an asbestos net when heating. The stir bar with a magnetic stirrer is of appropriate size and should not leak when poured.

The use of 　　 glass rod The glass rod is mainly used for stirring to make the solid raw materials dissolve in the liquid as soon as possible. Make sure that the glass rod does not touch the bottom or wall, and that the solution rotates completely and there are no dead ends. Some people think that the beaker will immediately melt and stir with a glass rod, but it actually breaks easily. In addition, it is very inappropriate to use a thermometer as a glass rod. use

　　 Magnetic stirrer One is to pay attention to the appropriate stirring speed, and the stirring rod should rotate effectively in the solution. The second is to pay attention to whether it is heated. There is no heating because it is thermally unstable.

Use of 　　 Funnel Certain reagents need to be filtered after preparation. When adding liquid, be careful not to drip the outside of the filter paper. Make sure to filter out small insolubles. use

　　 PH test paper Some solutions require you to adjust the PH value. PH test paper can be used in less demanding solutions. Pay attention to the pH range of the test paper. Then, once the liquid falls on the test strip, the colors are compared. Soak with a glass rod. After a test, the glass rod should be cleaned before use. use

　　 PH meter Use a pH meter to measure certain solutions that require a more accurate pH value. Pay attention to protect the electrode. When not in use, please use saturated potassium chloride solution to protect the electrode. In this regard, there is a special book for reference.

　　 Sixth, let’s talk about the use and storage of the prepared reagents.

　　 has already mentioned this issue when talking about storing raw materials. However, compared with the raw materials, the prepared reagents are usually liquids or suspensions and require more careful storage. As mentioned earlier, preservation is not discussed in detail here. Only mention places that require special attention.

　　A container of choice for reagent preparation. Normally, you will select all available containers around. Glass bottles are more commonly used. The smaller ones are penicillin bottles, also called vials. ** Doff type test tubes can be filled with very small amounts of reagents. The larger ones are 100ml, 250ml and 500ml infusion bottles. Please use as needed. Large bottles of distilled water are usually 1-5 liters, and in some cases 10 liters. The reagents can be packaged in bottles made of polymeric materials. This kind of bottle is usually used for toxic reagents. It can be discarded when it is exhausted. It looks a bit reluctant to look like a glass bottle. When you buy some kits, we usually provide some of these bottles. You may want to collect them, but they are useful in many situations.

　　Using other tools\nUsing a beaker Usually, a beaker is used for reagent preparation. Especially for reagents that require heating, a solvent (usually water) is usually added to dissolve everything, and then a solvent is added to supplement the water. Pay attention to the size of the beaker, and add asbestos netting when heating. The stir bar with a magnetic stirrer is of appropriate size and should not leak when poured. \r\nUse glass rods. Glass rods are mainly used for stirring, so the solid raw materials will dissolve in the liquid as soon as possible. Make sure that the glass rod does not touch the bottom or the wall, and that the solution is fully rotated and there are no blind spots. Some people think that the beaker will melt quickly and stir with a glass rod, but in fact it can easily break. In addition, it is very inappropriate to use a thermometer as a glass rod. Use\nFirst of all, the magnetic stirrer must pay attention to the appropriate stirring speed, and the stirring rod must rotate effectively in the solution. The second thing to pay attention to is whether it is heated. There is no heating because it is thermally unstable. \r\nUse of funnel Some reagents need to be filtered after preparation. When adding liquid, be careful not to let it drip out of the filter paper. Make sure to filter out small insolubles. \R\nSome solutions may require pH adjustment. PH test paper can be used in less demanding solutions. Pay attention to the pH range of the test paper. Then the liquid falls on the test strip and the colors are compared. Soak with a glass rod. After the test, the glass rod should be cleaned before use. Use\n PH meter: Use a pH meter to measure some solutions that require a more accurate pH value. Pay attention to the protection of the electrode. When not in use, please use saturated potassium chloride solution to protect the electrode. For this, there is a special book for reference. \r\nSixth, let's talk about using and storing the prepared reagents. \r\nThis issue was mentioned when talking about raw material storage. However, compared with the raw materials, the prepared reagents are usually liquids or suspensions and require more careful storage. As mentioned earlier, preservation is not discussed in detail here. Only mention places that require special attention. \r\nThe best container for reagent preparation. Generally, you will select all available containers. Glass bottles are more commonly used. The smaller one is a penicillin bottle, also called a vial. ** Doff type test tubes can be filled with very small amounts of reagents. The larger ones are 100 ml, 250 ml and 500 ml infusion bottles. Please use as needed. Large bottles of distilled water are usually 1-5 liters, and in some cases 10 liters. The reagent can be packaged in a bottle made of polymer material. This type of bottle is usually used for toxic reagents. Throw away after use. It looks a bit reluctant to look like a glass bottle. When purchasing a kit, you usually receive some of these bottles. You can collect them, but it helps in many cases. \r\nThe second is a label. Marking is a mandatory task. Some people like to write labels with markers. There are two types: oil-based pens and water-based pens, but the former is difficult to write and clean, while the latter usually disappear when wet. Write carefully with an oil-based pen. However, this method has its disadvantages. This means that next time you use this bottle with other reagents, the handwriting will not be easy to wash off and will be a little messy. You can also write on paper and paste transparent glue. Usually a wide adhesive is used so that it can cover the entire label and will not affect writing when wet. You can also write it on medical tape and paste it. However, this method may not be readable by water. You can choose these methods as needed. It is important to point out

　　The date must be on the label! In this way, you can know when to prepare reagents. If there is a problem, there is evidence. You can also let other users know if it is generally effective.

　　 The third is the accuracy of concentration. The concentration must be accurate, and some things need to be noted. The first is to choose the correct instrument. As with the reference solution, a volumetric flask is usually used, and an appropriate size must be selected. Equipped with general reagents and measuring cylinders. Never use a measuring cup. The measuring cup is a very rough guide. Pipettes* or microsyringes are usually used to prepare small amounts of solutions. Second, a very small amount (usually less than 5 mg) of reagent can be added directly to the storage container together with the solvent. For example, some reagents used for PCR and protein electrophoresis are almost invisible because they are only installed in a small amount in the Doff tube. Some require high-speed centrifugation before preparation, which results in a very small amount of powder deposited on the bottom of the test tube. Otherwise, once the lid is opened, the reagent powder will be lost. Third, ensure that the prepared reagents need to be cooled to room temperature and ensure that the final volume is accurate. For example, some reagents need to be heated to boiling during the preparation process, and a large amount of solvent will be lost, so other solvents must be added after cooling to room temperature.

　　4 is about special requirements. Some reagents require heating before preparation. For example, if the temperature of glacial acetic acid is lower than 16 degrees, it will freeze, so please heat it slowly before use. A similar phenomenon occurs in DMSO. Some reagents need to be shaded after preparation. You can choose black paper or black plastic package. Some reagents are not prepared with water, but require other solvents. Example: PMSF, prepared with isopropanol. And add to water when using. Because it quickly fails underwater. Certain reagents need to be filtered after preparation. Note the continuous filtrate. If the input is wrong, you need to repeat the filtering until the requirements are met. After prototyping, most of them need to be refrigerated and used as soon as possible. However, some may need to be frozen, depending on your specific requirements. Some reagents are easily oxidized, and antioxidants are usually added. Etc.: 6-hydroxydopamine (6-OHDA), usually prepared 0.2% vitamin C aqueous solution.

　　As a general rule, only some precautions should be mentioned regarding the use of reagents.

　　One is to use it as soon as possible after preparation. Since most of the prepared reagents are liquid, their shelf life is shorter than that of the starting reagents, and they are prone to failure. Therefore, you should plan your experiment before you start preparing reagents, and do not prepare reagents for a long time before you start. Second, when reusing reagents, care should be taken not to contaminate the reagents. When removing liquid by pipetting*, please be careful* to replace the head. When pipetting, wash carefully and discard the excess liquid without returning it to the bottle.

　　 Third, do not use different batches of reagents in the same experiment. This is because the focus of animal research is to compare the same things. When using different reagents, the reason for the difference is unknown. Therefore, when preparing reagents, try to dispense as much reagent as possible. Try to run the excess reagent in an experiment instead of running out.

　　 Fourth, if you want to use reagents prepared by others, you need to know the source and related information. When conducting the same type of experiment in the same laboratory, they usually share several reagents. At this point, you should pay attention to asking the question clearly. Otherwise, if there is a problem with the experiment, it will cause great losses. For example, when performing protein electrophoresis, the buffer used, ammonium persulfate and acrylamide are the same, but the ratio is different. Be careful not to use expired reagents. Fifth, the relevant cooking utensils must be cleaned after the reagents are used up. Some may need to be cleaned immediately after use to prevent others or themselves from being contaminated and misused. Etc.: Toxic acrylamide solution. Some people wash all the tools in a short time. The rest of the liquid is not cleaned, so it is difficult to clean for a long time. This is something we should pay attention to. Please be aware of this, as some laboratories may have to separate equipment. It's like washing dishes after eating. This should also be a good experimental method.

　　 Seventh, let’s talk about common problems and solutions in reagent storage.

　　 During the storage process, the prepared reagent has some problems. Some can be treated a little without affecting the use, while others may not be used continuously and may even cause toxicity. Therefore, understanding the general phenomenon of the reagents that may degrade will help you understand the general laws and solve experimental problems smoothly.

　　 physical property changes

　　 The most common are precipitation or crystallization, flocculation, color change, viscosity change, mold, and odor

　　 Precipitation or crystallization: The most common is certain buffers.. If the temperature is relatively low, some substances may be saturated solutions, and the low temperature may cause them to precipitate. It will dissolve when returned to room temperature. For example, when I try to make a solution containing multiple zinc chloride ions, precipitation appears when I open the refrigerator, and when shaken at room temperature, it is transparent and does not interfere with use. Improper preparation of Coomassie Brilliant Blue may result in blue precipitation. Generally speaking, avoid proper adjustment of ethanol concentration.

　　The formation of flocculant: some substances will precipitate on the flocculant at low temperature, and usually will not affect the use. However, please note that if the flocs are other substances or mycelium, they cannot be used. Be especially careful with aqueous sugar solutions.

　　 Color change: This change may occur after heating for a period of time during the preparation process. It may also be caused by exposure during storage. It may also be caused by the interaction of various substances in the solution. The color is usually darker. For example, when I prepare a new compound, I don't know it in advance, so it will melt immediately after heating it, and when I put it in the refrigerator, it will turn yellow the next day. When I asked the people who synthesized this compound, it was unstable above 60 degrees. In addition, the color may fade. Etc.: Some dye solutions. Viscosity changes: "solutions" of certain polymeric substances. In fact, the polymer is dispersed rather than truly dissolved. Waiting for the gelatin solution, if the preparation time is too long, some may become thin, which may cause problems in the quality of the gelatin.

　　 Mold production: It is common in solutions containing high sugar water. The main reason is improper storage. Generally, pay attention to operation during preparation. After cooling the preparation, it will not form mold in a short time.

　　 Odor: This is mainly due to the fact that the substances in the solution react with each other to produce gas, the dissolved gas disappears, and the inherent odor changes. Like: produce ammonia smell, lose acetic acid smell, etc.

　　B. Changes in chemical properties

　　Example: PH value change, loss of oxidation, loss of reduction

　　 PH change: If you suspect that there is a problem with the reagent, you can measure its pH. If it exceeds the normal range, it is considered degraded. Reduced oxidation: When using concentrated sulfuric acid to measure the polysaccharide content by the anthracene sulfate method, it was found that the prepared reagent did not react and showed no color. I dropped a drop of concentrated sulfuric acid on the newspaper and found that the newspaper was not carbonized as usual. There is currently no such sulfuric acid. A simple observation can determine whether the reagent has deteriorated. Similarly, certain strong oxidants, such as hydrogen peroxide, can be judged by similar tests.

　　 Reduce reducing performance: reducing substances such as vitamin C usually change color when it deteriorates.

　　C changes in biological characteristics

　　 Examples: enzyme inactivation, biomolecule degradation, etc.

　　 This change does not appear immediately. When the results are unsatisfactory during the experiment or after the experiment is completed, reagent problems usually occur. This issue will be discussed in more detail in the chapter "How to track reagent problems in an experiment". I will not repeat it here.

　　How to solve the problem of reagent storage?

　　 Generally speaking, in addition to some phenomena that do not affect the use of reagents, reagents need to be reconstituted.

　　 Eighth, let’s talk about the preparation and storage of toxic and dangerous reagents.

　　 First, let us understand the basic common sense of toxicity. According to the length of action, it can be divided into acute toxicity and long-term toxicity. According to the distribution of its effects, it can be divided into local toxicity and systemic toxicity.

　　 According to its location and mechanism, it can be classified as follows.

　　 Organ toxicity: For example: chloroform has liver toxicity; SDS powder can be deposited in the lungs;

　　 Blood toxicity: etc.: Benzene has blood toxicity

　　 Neurotoxicity: etc.: Acrylamide is neurotoxicity. Carcinogenicity: etc.: Ethyl bromide can cause cancer, and urethane can cause cancer.

　　 Other toxicity: allergies, skin irritation, etc.

　　 It is very important for us to understand the toxicity mechanism. In this way, we know how to protect and dispose of toxic reagents.

　　 I don’t care much about the local or chronic toxicity of certain organs. But because of the toxicity that can cause blood, nervous system and carcinogenic toxicity, this is the most terrible thing for us. In fact, as long as you are safe. Some carcinogens should also be used with caution. Our body is not so fragile, our immune system is working.

　　 However, carrying out the necessary protection work is the most important.

　　 Let us talk about how to protect ourselves during the preparation of toxic reagents.

　　1 To understand the toxicity mechanism of the reagent. This is the basic premise of protection. Usually labeled as a toxic substance, the mechanism is almost clear. In this way, final conclusions about toxic agents can be drawn. It has been found that long-term use of solvents such as chloroform can cause fatty liver. However, when you use it, you just need to sprinkle it on your skin occasionally for care, so don't worry. If the concentration of hydrogen peroxide is not high, it will not cause much damage even if it adheres to the skin, but it may fall off slightly. But there is no danger to life. On the contrary, if you drop a drop of carcinogen, you will feel scared. Therefore, to understand its toxicity mechanism, we know what a reagent is, and don't need to care or worry about it too much.

　　2 In order to understand the nature and precautions of commonly used poisons, different measures have been taken for different poisons. For example, poisons are volatile substances. Put on a gas mask, prepare to ventilate and cover it tightly. For substances that easily bring dust into the lungs, wear a muzzle and keep it as light as possible; be careful; for substances that may cause cancer, please wear disposable gloves..

　　3 10,000 people are not afraid to know the symptoms of poisoning, just in case. It is usually impossible to avoid 100% exposure to toxic substances. You need to know some symptoms of addiction. Such as: redness, pain, eye irritation, some bad smells. If it gets on the skin, rinse with plenty of water. In severe cases, please go to a clinic or hospital for treatment.

　　 Based on the above knowledge, let us talk about the precautions for preparing toxic reagents.

　　 First of all, the container for the toxic reagent is dedicated and requires a striking logo. For many toxic reagents, we use polymer bottles unless glass bottles must be used. Since glass bottles are always reused by people, it is possible to misuse bottles containing toxic reagents without anyone knowing. Red labels are usually used to indicate "Toxic!" and add toxic symbols as needed.

　　Second, prepare for the protection of toxic reagents. First, you usually need disposable gloves and wear a mask and gas mask as needed. Second, the reagent should not touch the outer wall of the container. After the reagent adheres to the outer wall, disposable gloves must be worn after each use. In this way, disposable gloves with toxic reagents may accidentally touch other bottles, thereby increasing the degree of contamination. Our method is usually to replace the bottle immediately when the outer wall is contaminated. In this way, you can directly hold the bottle wall in your hand without worry. When unscrewing the lid, usually the lid should be facing up. This can also reduce the degree of pollution. Third, once it is ready, the pollutants will be delivered to the designated location in time. Including bottles containing toxic reagents, gloves and other disposable equipment. Fourth, equipment exposed to toxic reagents for a long time should be placed in a dedicated area. For example: the electrophoresis tank used for PCR, long-term exposure to the electrophoresis solution, the area of EB etc. should be fixed within a certain range and should not be placed anywhere. This will minimize pollution.

　　 Third, the preparation of toxic reagents should be carried out in accordance with procedures. Pay attention to reagents that react with other reagents to produce carcinogens. Example: Diethyl pyrophosphate (DEPC), a reagent commonly used in PCR, reacts with ammonia to form carcinogens. However, it is usually used to soak and volatilize chip heads. Reagents containing Tris and DTT cannot be treated with DEPC because DEPC can react with amines and sulfhydryl groups. In fact, here are some potential carcinogens. Please pay attention to contraindications during use.

　　 Let us also briefly explain the preparation of dangerous goods reagents.

　　The dangerous goods mentioned here mainly refer to substances that may explode under certain conditions. Wait:

　　 Hazardous substances, such as ether and aniline, are usually used under low temperature, shading and specific conditions. Pay attention to the conditions causing the explosion.

　　 Ninth, let's talk about the optimization of preparation reagents.

　　Usually, we always strictly follow the steps in reference books, literature or reagent instructions to prepare reagents. However, these steps can be tricky. So what do you think of improvement? How fast! What are the requirements for reaching standards that seem to be higher than the average experiment?

　　 First, you need to fully understand the principles and potential problems of the reagents.

　　 Next, some elements need to be improved in the experiment.

　　 Third, you need a small amount of trial experience. Method improvement is not a one-time exercise, but a continuous iterative research. preparation

The main optimization method of 　　 reagent is as follows.

　　1 When mixing reagents and using reagents for detection, multiple reagents must be prepared, and multiple reagents must be continuously and continuously added to the sample. First of all. Due to the error of addition, the ratio of various reagents may be different each time. Some reagents, especially those used to detect enzymes. However, if you understand these principles, you can create hybrid solutions and add them all at once. For example, in an enzyme reaction experiment, the substrate and its buffer can be several reagents, but usually they can be added as a mixture before adding the enzyme. This can reduce errors on the one hand, and save time on the other. 2 Appropriate concentration change reagent manuals and procedures for specific experimental methods usually describe the concentration of reagents that should be prepared. But I am not saying that this cannot be changed at all. In some cases, due to different experimental conditions, reagents may be prepared according to the methods in this book, and experimental results may not even be obtained. So under what circumstances can the concentration of the reagent be changed? In other words, why not make changes yourself based on books or literature? Suppose you need to understand the principle of the method. For example, when performing protein electrophoresis and staining with Coomassie Brilliant Blue, it may not stain. At this point, you can consider increasing the concentration of Cooma or loading more protein. Another example: giving an animal an animal indicates that each animal has a certain ability. And our dosage is different. Therefore, you need to adjust the density for different situations and do not copy the density into books or literary works. In addition, when measuring the content of bear-like protein, it is sufficient as long as the ratio of bear to protein is a certain amount. If the concentration is within the detection range, insufficient reagents will reduce the protein content of reagents and analytes by 2/3, and even measure protein content. These are all different. Therefore, if you are familiar with the principles and procedures of the experiment, you can learn the experiment without relying on this book.

　　3 steps have been omitted appropriately. For solutions with incorrect concentration requirements, some steps can be skipped in the preparation process. This varies from person to person. I think there are many such situations in the experiment.

　　4 The prerequisite for the improvement of reagent prescription is to have a full understanding of experimental principles. The formula of Coomassie Brilliant Blue has been changed. When I first started protein electrophoresis, I used molecular cloning recipes for preparation, but it was not always stained. It's strange that after a period of exploration, I think this may be Cooma's problem. The loaded protein mass is large enough to increase. So I changed Kumar's formula myself. As long as the proportion of methanol in the Kuma liquid increases, it proves that it is a fouling solid. Therefore, according to the original recipe, we prepared several different Cooma solutions and tried some adhesives. Choose a concentration that will not only stain the color, but also distinguish the depth of the protein band. This is the highest concentration in my experiment. In many cases, laboratory conditions will be different and the reagents used need to be improved. This book is only a rough standard. It is very flexible.

　　5 The alternatives used in the experiment to find alternatives may not be timely. Some were given by others, and some were mailed. However, some less commonly used reagents will actually be lost for some time. So should other reagents be replaced based on experimental principles? I think you can think of it. However, it is basically assumed that the experimental results will not be affected. This requires very proficiency in the experiment, and familiar with the specific experimental procedures and principles. Normally, this method is not recommended.

　　 Tenth, let's talk about how to track the problems caused by the reagents in the experiment.

　　 Sometimes it may not be immediately obvious that there is a problem with the reagent. Remember that even if the results of the experiment are abnormal, there may be problems with the reagents. In animal experiments, the earlier it is discovered, the more suitable it is for experimentation. Waiting for the experiment to complete and checking it is usually useless. Animal experiments are sometimes difficult to repeat. What we can do is to use the same batch of reagents in our experiment and compare all animals. Only in this way can you obtain reliable information.

　　 First, let's talk about how to avoid the experimental problem of reagents.

　　 is to ensure the reliability of reagent materials. You need to use imported. It is best to go to a large, well-known company. Don't buy money-saving products that are difficult to meet the requirements. For example, the acrylamide and methylene contained in protein electrophoresis reagents can be imported in original packaging or in imported quantities, but domestic products are not recommended. Some reagents made in China may not meet the requirements. Of course, not all reagents need to be produced in China. This largely depends on the experimental requirements and quality standards of household reagents.

　　Secondly, do not frequently change the brand or manufacturer of the reagent. In the exploratory phase, you may want to try various reagents that you know. However, it is basically necessary to follow up which reagent is used. Especially in the large-scale experiment stage, in order to ensure the same results in the experiment, please do not rush to buy the required reagents and lose them all the way. Not only reagents from different manufacturers are different, but different batches from the same manufacturer may also be different. Special attention should be paid to biological experiments.

　　 Third, try to avoid contamination during reagent preparation. One is microbial contamination, and the other is other impurities. The most demanding reagents are usually special bottles. When changing reagent batches, the last batch should be cleaned up. If possible, try dedication. If you want to share reagents, please provide them with detailed information or get detailed information from them. In short, please feel free to use reagents.

　　 Fourth, prepare reagents and store them as required. This is not very obvious. For example, *** can be a *** solution for animal marking, or it can be Bouin's solution. Both look the same color. Write a good logo to distinguish it. Another example: PMSF is formulated with isopropanol and stored at -20°C.

　　5 is to pay attention to the operating procedures of reagent use. Usually, the laboratory will create the same SOP to use common reagents. Some reagents have detailed instructions. I don't think everyone is wrong. Before use, detailed inquiry is the most basic warranty.

　　 However, even if such precautions are taken, it is difficult to avoid certain problems in the experiment.

　　 In fact, because animal testing is biological, the biological variability is too large and some problems inevitably arise. What we have to do is how to investigate the experiment to find the root of the problem.

　　There are several ways to find the problem in the experiment. One is the daily inspection method. This method confirmed the basic and simple chemical properties of the reagents used in the experiment. Example: The reagent is already liquid at room temperature, do I need to freeze it? Is the PH value within the specified range? Has the reagent been removed by visual inspection? Will the color change? Is the measuring instrument clean? Have you used expired reagents? wait. These tests will give you an immediate understanding of why the experiment failed. However, many of them cannot be found at once. This requires a step-by-step investigation using some skills or means. The second is the replacement method. The way to do this is to reconstitute the reagents in a specific step of the experiment, or run the reagents with the same reagents prepared by others to confirm the results of the experiment. The premise of this method is that some reagents have major problems. Example: If you suspect that there is a problem with the cell lysate during protein electrophoresis. You can rewrite it or try it with others. So you can find some information. This is the most common way to resolve the cause of experimental failure. The third method is reinforcement. The so-called enhancement method is one of the magnification factors to see if it is caused by insufficient reagents or insufficient sample volume. In this way, specific dosage factors can be highlighted to reveal reagent problems and eliminate factors. Example: Protein electrophoresis is not stained. I want to know if it is due to lack of samples. At this time, increase the sample size. Usually 50 ug, research experiment can be 200, 300 ug or more. If the color does not change after increasing the number, the adhesive surface may become dirty or dirty. At least this may not be the reason for the small sample size. Based on this, you can further investigate the reason. 4 is G