 Dear students, today we will learn about preparation of stocks and working solutions. It seems to be basic but actually an important task while working in the laboratory. A solution is a mixture of one or more solutes in a solvent. Before going to preparation, I would like you to introduce about certain precautions and procedures. For example, while measuring the liquid reagents, we should use some narrow neck apparatus like measuring cylinders and they should be properly labelled with a tag which is mentioning the name of reagent, date of preparation of that solution, the initial of the person who prepared these solutions, lot number and expiry number. Dear students, solution preparation is very important task while working in the laboratory. Now we are going to have some introduction about solutions and their types. So there are two components, solute and solvent. Solvent is a medium in which solute is usually dissolved. Briefly, such as solutions can be of different types depending on the concentration of the solute they have. So based on the concentration of the solute of different components of the of solutions, we have various types. For example, molar solution which is represented by capital M, molar solution which is denoted by small m, percentage solution and part solution. They can be parts per million, parts per billions but we will only talk about parts per million which is denoted by TPM. Further percentage solutions can be divided into weight by volume, weight by weight, volume by volume and weight by volume. How to calculate the concentration of different solute in these solutions? This varies. First of all what is molar solution which is denoted by capital M? It is number of moles of solute in one unit liter of solvent. So number of moles of solute in one unit liter of solvent, there is a formula for that. Mass of solute in grams can be calculated using formula weight of solute multiplied by the molarity required multiplied by volume we need to prepare for that solution. The final volume we need to prepare for that solution. For example, if I say that I need to prepare one molar solution of tris buffer and it is 0.5 liters. So I am going to put the values in the formula. So how much tris base we are going to measure to dissolve in a solvent? That will comes by using this formula. Formula weight of tris base is 121.1 multiplied by molarity we need one molar of the solution. So we will put one. Then we have to add the volume in that formula and the volume we have is 0.5 liters or you can say 500 ml. By putting in the formula we will get the answers in grams which is about 60 grams. So we will weigh about 60 grams and dissolve this 60 grams in final volume of 0.5 liters. We will get our solution. It is noted by small m and in this we need to have number of moles in per kilogram of the solvents. That means Kappa solvents is measured in kilograms. As usually we will use molar solution and the rest of the solution we are going to prepare for this experiment is that they are molar. So I have given you the tail of molar solutions. Next is the percentage solution. Percentage solution as we already talked about they can be weight by weight. That means that the solvent as well as solute both are in. We will measure them in weight or mass. They can be volume by volume both will be weight in liquid volume. But the most common we will use is weight by volume that the solute is in powder form in solid form and the solvent is in liquid form. So for calculation of percentage solution of weight by volume we can take just the example of 10 percent STS. STS is basically Sodium Dodicisulphate is one of the solutions that we use commonly in the lab and we will also prepare it. So to prepare 10 percent weight by volume STS solution that means we have to take 10 gram of STS powder and make the final volume up to 100 ml. But by resolving 10 gram of STS in a final volume of 100 ml we can get 10 percent weight by volume STS solution. This is about the brief introduction of the types of the solutions that we commonly use in the lab. Now there is another terminology that would I like to introduce you about. It is about stock solution and working solution. The solution we have in laboratory they are of you can say two categories stock and working. As the name show stock is something that has high concentration and it is useful for storage and further use and working is something that has the measurement of our required experiment according to required measurements according to experiment. Most commonly used purpose for in cell or molecular biology laboratory are stocks are 1 molar tris, buffer, 0.5 molar, ADT buffer, 5 molar, NSEL buffer. In addition to this often we also make a concentrated solution to save storage space as well as to spare to preserve the time. How you can prepare a working solution from stock solution. This is very important formula that you should be going with and this is C1 V1 is equal to C2 V2. C1 and V1 represents concentration of stock solution while V1 represents volume of stock solution. Two represents working so C2 stands for concentration of working solution and V2 stands for volume of working solution. The most common example of stock and working solution that are used in molecular biology laboratory is 50x electrophoresis buffer. 50x means 50 times concentrated and the buffer name is TAE. This buffer is used for electrophoresis. We have this buffer in our laboratory in stock and but while working in the lab while running the gel buffer we need it 1x TAE. How we are going to convert your calculate for the calculation of 1x TAE. So we are going to use a formula C1 V1 is equal to C2 V2. For example if I provide you a situation that we need to prepare 1 liter of 1x TAE buffer. I will give you a flask or beaker and I will say you that dear students go and prepare 1x TAE buffer and I will provide you an empty buffer, empty flask and another flask which is having the stock of stock solution of 50x TAE. How you are going to prepare 1x TAE buffer from that. For this we have to use the formula we know the concentration of stock buffer is 50x. So instead of C1 we are going to put 50x here multiplied by volume. How much volume we will be needing to be drawn from 50x and to add in 1x it is a question mark. This is the volume which we need to know which we need to calculate. Question of our working which we need to make is 1x so we will put 1x here while volume of working solution is 1 liter. So we will put 1 liter here. By slide adjustment the question mark which is actually the volume of stock needed to be added to make 1x TAE can be calculated by multiplying 1x with 1 liter and dividing with 50x. This 50x comes below the to the equation. So we will get the volume and the desired volume that will come here is about the brief introduction that how there are different types of solutions and how we can make working solution from the stock solution. Now we will go towards the preparation of the solutions. Dear students most commonly for preparation of solutions whether it is stock solution or working solution we need some instruments like wing balance magnetic stirrer or we can also say hot plate and pH meter. Let me introduce you about these instruments a little bit. So first of all wing balance is an instrument which is used to measure mass of an object. There are different types of wing balance that are available but usually they are of two type. One is analytical and one is precision wing balance. They both vary in the quantity they can measure as well as in the precision in that wing measure. Analytical balance usually has different windows so we can measure from different dimensions like you can add things from here or you can use that window. So this analytical balance first of all this is a sign of power button from where we can switch on the balance for they are certain instructions for putting this analytical balance in a lab for example it should be on a balance surface as well as it should not be in direct contact with the air otherwise the powder or the mass you are going to measure can be interrupted. So you can see this wing balance can measure up to 0.000 grams so it has high precision value this is analytical balance. So whenever you measure something you need to tear down or zero the weight of your container or wing paper. So after putting the wing paper you need to tear it so that the volume or the mass of the wing paper is become zero. This is magnetist error also called as hot plate magnetist error comes in different dimensions and different models they are some which are also digital they are some with function of heating this magnetist error has two functions stirring and heating so whenever you need to make something you need to add a magnet a vice flashes put your container on the plate this is a plate which has magnetic field which allow this magnet to stir switch on stirring you can adjust the speed as you require. This hot plate also has a function of heat so you can also adjust the temperature Some types of strutters comes with different range of heating but usually they are they can go up to 70 degree centigrade from room temperature pH meter is a very sensitive instrument and usually it is used to measure the pH of the solution it has certain parts these can come in different models different displays some are digital some are buttons but mostly they have three common parts one is a display or meter one is arm you can move this arm so that you can easily go to your solution and third is the most sensitive part which is probe so for each time you use the probe of pH meter it is needed to wash it for washing remove the probe from the storage buffer usually the probe is stored in a case here solution put the probe in a container in a in which you want to wash it this is the bottle with distilled water and you can wash the probe up to down with distilled water now you can use this probe to your required solution and you need to each time before and after using this probe to wash it with distilled water for preparation of one molar crisp buffer pH 8 we need to take distilled water for 100 ml buffer initially we take some less amount of water like 80 ml water and put it in a glass beaker crisp base we will add 12.1 gram of this base but before adding the powder we need to balance out or tear the weight of this paper and now we will add some amount in this then put this describe amount in the beaker and put the beaker on the stirrer we will switch on this and keep stirring until it clears or dissolve all the powder after the solution of the complete powder we will check the pH of this solution by using pH meter before adding into the solution we need to wash the probe the pH of crisp solution should be 8 for using this we will need to add acid in that so we will add we are going to add acid in this until its pH becomes 8 after completing the resolution of the powder and setting of pH we will now make the final volume for this as we are preparing one molar crisp solution of 100 ml we will put all the volume back in the slender then add water to make it 100 ml now we need to store the prepared solution in a reagent bottle clean at its appropriate storage conditions this is one molar crisp solution pH 8 now we will prepare 0.5 molar edta solution for preparing of this solution we are going to prepare this for 100 ml buffer after making the amount we are going to add this in this to water to dissolve it add a magnet edda is highly acidic it would not clear until its pH becomes 8 as you can see its pH goes in the acidic range we are going to add NaOH in it drop by drop let it dissolve okay after the buffer solution pH is 8 you can see that its color becomes clear now finally we are going to make the volume up to 100 ml with the help of a my ring slender okay now this is 0.5 molar edta solution 100 ml has been prepared we can store it in a clean reagent bottle now this stock solution can be prepared can be used for few months if stored on appropriate conditions now preparation of five molar sodium chloride solution five molar sodium chloride solution preparation we need sodium chloride powder and we are going to make 100 ml of dissolution so we are going to add less than 100 ml amount of distilled water in a beaker i am taking about 70 ml of distilled water and in that amount of water we need to add 29.2 grams of NaCl solution powder so as you know that we need to tear off the weight of paper first when it shows 000 we will add NaCl powder 29.6 gram NaCl powder should be added into approximately 70 ml distilled water which we have measured earlier then add magnet in it this five molar NaCl solution is basically a super saturated solution that's mean we need heat to dissolve it as you can see that the solution's appearance is turning transparent as heat is going to dissolve the powder five molar NaCl solution has become transparent after dissolving it and hitting it so we are going to make the volume final by adding back into the cylinder as we are making it 100 ml so we need final volume up to 100 ml as you can see certain volume has already raised because of the powder of NaCl we have added as you know for transparent liquids we use lower meniscus to have accurate volume now this 100 ml five molar NaCl solution is completed we can store it in a clean reagent bottle and use our in our further experiments now we are going to prepare 10 percent sodium toticisal fate STS solution weight by volume for preparing this first we are going to weigh the 10 grams of this STS powder so before weighing we need to tear down your zero the weight of paper and then add STS STS is slightly irritating to the mucus membrane so you should take appropriate precaution while handling with the STS powder now put the powder in a beaker and we are going to add some distilled water in it we need 100 ml volume but initially we are going approximately 70 ml of distilled water this STS has surf like properties so try not to shake it otherwise foam will appear we are going to add magnet in it and turn on the stirring of the hot plate as well as we need some heat so that STS can dissolve easily you will see there is certain form is appearing and initially the color of this mixture is turbid but as it dissolves it will appear slightly transparent as you can see this 10 percent STS powder has been dissolved as you can see the appearance of the solution is transparent now we will switch off the stirrer as well as heat and make the final volume 100 ml we will add the dissolved solution in a measuring cylinder and make the final volume up to 100 ml now this 100 ml STS solution has been prepared and we need to store this in a clean reagent bottle now this 10 percent LTA solution has been prepared you can use it whenever you are required students the next practical solution is the normal NOH solution you know NOH is a basic and usually NOH is found in a form of pellets so first of all as you know we are going to weigh our NOH pellets we need 40 grams of NOH to make 100 ml solution so I am going to tear or zero the weighing paper when the reading is zero we are going to add our pellets to make 40 grams of the weight NOH is highly basic so you should be careful while handling its pellets and we will be needing distilled water so I am taking less than 100 ml of distilled water I am taking about 65 ml of distilled water because when we are going to add the NOH pellets the weight is going to add up to clean beaker add distilled water and add your weight NOH pellets in that beaker now you need to turn on the stirrer it may take a while for the pellets to be dissolved you will also notice that heat is produced when these pellets are going to be dissolved so don't turn on the heat just turn on the stirring function of the hot plate as you can see whenever you store a reagent the region bottle should be properly labeled as I mentioned earlier with the name of the solution date of preparation it's initial of scientist who prepared a solution lot number and its expiry date so it's preferred to clean the reagent bottle beforehand and label it properly mostly this highly basic solution is used to for ph measurements or to adjust ph of other solutions after complete resolution of the pellets of NOH we are going to turn off the stirrer and put the solution in a volumetric cylinder and finally make the volume of 100 ml as we have added the NOH pellets for solution of 100 ml so it's about 70 ml you need to add some more water distilled water be careful about the measurement for small volumes we can also transfer water using the pipette now this is 100 ml complete we are going to add this in a reagent bottle the reagent bottle should be clean and the solution can be stored at room temperature for few months now the normal NOH solution is ready students now we are going to prepare 10 milligram per embers ethyl mermide solution ethyl mermide basically is a chemical that is used to strain DNA while running in a gel electrophoresis procedure so for this we need 0.2 gram of ethyl mermide powder this powder is carcinogenic because it can bind to DNA so you have to be very careful while handling this powder i have already measured 0.2 gram of ethyl mermide powder and we are going to add this powder in 20 ml of distilled water because this powder is very dangerous so i'm not going to add this solution in a cylinder we will directly measure 20 ml water and add 0.2 gram of ethyl mermide mix it well it might take some time for mixing as you can see it's slightly orange color it gives those DNA orange color while we see the DNA in UV light after proper mixing the storage condition is very important because we have to store this ethyl mermide in dark for that it is recommended to use dark bottle or you can cover this with any sheet that can reflect light for example we can use aluminium foil to cover this falcon as light can degrade this chemical so this is your 10 milligram per ml ethyl mermide which is ready it is recommended to store it at 4 degree centigrade for further use dear students the next solution preparation is about 50x ta solution that is used for electrophoresis for this preparation we need 24.2 grams of crisp base so we are going to measure the crisp base powder then we will add 5.7 ml of acetic acid and 10 ml of edta 0.5 molar solution so first of all we are going to put our weighing paper on the balance tear it when it becomes zero we will add crisp base powder equivalent to 24.2 grams the desired amount of crisp base should be added in a beaker and we will add some distilled water in the beaker so that crisp base can be dissolved and as we are going to prepare 100 ml of 50x ta so i am going to add some less amount like around 65 ml of water distilled water in it turn on the stirring for crisp base to be dissolved so we have already prepared 0.5 molar edta buffer pH 8 using a pipette carefully take 10 ml of edta buffer and add in the crisp base the third ingredient is glacial acetic acid as the name show it is acid so you should be very careful about handling this acid we need 5.7 ml of this solution you can take this through a glass pipette or you can also measure using a micropipette we need 5.7 ml so i am going to add 5 times 1000 microliters that would be equal to 5 ml then i can change the measurement to 700 microliters finally adding this amount discard the tip of acetic acid carefully as you can see the whole ingredients have been dissolved we will turn off the stirrer and make the final volume of 100 ml for that we will use graduated slender and we need to need to make the final volume of distilled water up to 100 ml after making the final volume we need to store the 50x buffer in a clean glass reagent bottle so our 100 ml 50x ta buffer has been prepared you can store it at appropriate conditions and use it for few months dear students now we are going to prepare 2x gel loading dye this dye is used for a gross gel electrophoresis for separation of different size of DNA molecules to prepare gel loading dye we need three ingredients first of all 2 percent bromophenol blue 2 percent xylene cyanol and glycerol xylene cyanol and bromophenol blue are basically dyes that give some colors to the sample i am going to prepare in a falcon so i am going to add bromophenol blue which is 2 percent already prepared solution 250 microlitre so on the pipette i have already said 250 microlitre we should be careful about by using the dyes because it can stain your garments now the same amount about 250 microlitre of xylene cyanol which is 2 percent while running on the gel you will see blue color of this dye under the influence of electricity running through the gel and last ingredient is 7 ml of gristrol gristrol serves true purpose while used in the gel loading dye gristrol gives density to the sample so that if the DNA sample will go straight into the well mix it by inverting several times and your 2x gel loading dye for a gross gel electrophoresis is ready this will be mixed in your DNA sample while you are going to run the gel students the next particle is about preparation of 1x ta buffer basically 1x ta buffer is a working solution that we are going to prepare from 50 x ta buffer the calculation at how much amount of this 50 x ta buffer will be added for to prepare 1x ta buffer can be obtained from simple c1v1 is equal to c2v2 formula that has been explained earlier so according to the formula we need 10 ml of 50x to make final volume of 500 ml we are going to measure 10 ml using pipette you can also use small slender graduated slender to measure 10 ml volume carefully transfer 10 ml of 50x ta buffer in a reagent bottle now we need to make the final volume up to 500 ml as we have added 10 ml so i am going to add 40 ml distilled water plus 450 ml of distilled water that will give us a final volume of 500 can close it mix it and 1x ta buffer is ready to be used in a gel apparatus you can store it at appropriate conditions dear students te buffer is our next solution that we are going to prepare in this solution pre stands for pre-seal buffer and e stands for edt buffer so we have to mix these two buffers in a certain quantity we have already prepared 0.5 molar edt buffer and one molar dissolution buffer for preparation of 100 ml of te buffer we need 400 ml volume of edta so we are going to measure this with the help of a micro pipette this micro pipette can measure up to 1000 microlitre but we are going to use 400 of it so 400 microlitre of 0.5 molar ph8 edta and 1 ml of 1 molar tris buffer but before taking the tris buffer we need to adjust the pipette up to 1000 microlitre this tre buffer is often used for washing of cells and it's also used for storage of biomolecules like proteins and eplic acid now we have to make the final volume up to 100 ml this tre buffer is ready and we need to store it in a clean container appropriately labeled so this is tris edta buffer ready to be used students the next practical or preparation of solution is about te and buffer te and buffer is often used for extraction dn extraction practical in which t stands for tris e stands for edta and n stands for nacl so these are three basic ingredients we have already prepared the stock of 0.5 molar edta stock of 1 molar tris solution and 5 molar sodium chloride solution let's do the preparation i'm going to mix all the ingredients in a graduated cylinder first of all we need to add 1 ml of 1 molar tris buffer to measure this i'm going to use a microfiber which is already a stage at 1000 microlitre equivalent to 1 ml carefully measure 1 ml tris solution we need to add 0.5 molar edta buffer equivalent to 0.4 ml next or third again dn is 5 molar sodium chloride solution and we need to take it 8 ml for measuring this i am going to use pipette so 8 ml of sodium chloride solution should be dispensed into the container after adding our three ingredients 1 molar tris base 0.5 molar edta and 5 molar sodium chloride solution we need to make the final volume up to 100 ml and now this solution is ready store this solution in a clean labelled virgin bottle and this t en buffer is ready to be used