REAGENT ACQUSITION AND STORAGE You decide you want to try a particular experiment.An idea you had.An idea you got looking @ the literature.An idea you had with your colleagues.Etc.,You determine what reagents you will need to perform your experiment.If you do not have all the reagents you must therefore obtain any missing (reagents).QUESTION # 1How much reagent do I need?To answer this question, you must first determine:How much reagent is used per rxn?How many rxns in total per experiment?How many experiments do you intend to run?If you are duplicating a rxn in the literature, you use the amount given per rxn times the # of desired rxns times the # of experiments you plan to run.You, thus order at least a little more than this total.For example, if the literature called for 5 M ACTH1-39 in a 50 L final vol and you want to run 10 rxns per experiment and you wish to do 2 sets of experiments, you would need:(50 L /rxn)(5 mol/106 L)(103 nmol/mol)(10 rxns/experiment)(2 experiments) ≥ 5 nmol ACTH1-39 total.The reagent concentration per rxn may be given in international units (I.U.), radioactivity,weight or mass per unit volume, weight or mass per unit mass, etc, In these cases, you may need to convert to the relevant units to perform the necessary calculation(s).For example, the MW of ACTH1-39 = 4567 Daltons. 1 Da = 1 gThus, 1 mol of ACTH1-39 weighs 4567 grams.1 mol = 6.023 x 1023 molecules.You may thus need to convert to the appropriate units. 1REAGENT ACQUSITION AND STORAGE Now you know how much reagent you will need initially.QUESTION # 2What increments are available for purchase of the desired reagent?To answer this question, you must:Look to the manufacture.Compare different manufactures.Pick one.QUESTION # 3How much reagent to order?To answer this question, you must consider:Reagent cost.Reagent stability.You find that no one sells the small amount necessary for your preliminary experiments.Thus, you decide to buy the least amount available for purchase among the relevant suppliers.Manufacture reliability, recognition, availability and consistency are important considerations. (The right tool for the right job).The reagent arrives and you store it according to the manufacture’s recommendation until ready to use. For example, it might state to store < -20oC, light-sensitive, desiccate upon opening and avoid repeated cycles of freezing and thawing. Thus, you put the reagent into the freezer in a lightproof container and wait to desiccate until opening.QUESTION # 4How do you determine an aliquoting strategy?To answer this question a variety of factors must be considered.Reagent stability is of major importance. Generally, if you do not need to compound a substance, it may not be necessary to prepare it in advance. In fact, some reagents MUST be compounded fresh before each use. On the other hand, 2REAGENT ACQUSITION AND STORAGE you may want to make as much as reliably possible if you are continually using a reagent. Often times, it is practical to generate aliquots of reagents under different physiochemical conditions for storage or use under different conditions. Length of storage until use, pH, molarity, cofactors, etc., all enter into aliquoting strategies.N2 (l)  -150oC DMSO; 50% glycerol  -135oC - -80oC;Nothing (possibly a preservative, e.g., sodium azide) –20oC, 4oC, R.T. Useful resources for proper reagent conditions (i.e., storage and handling).MSDS, Product Spec Sheets, The Merck Index, Manufacture’s Tech. Support, relevant Literature.Compounding stock solns.Sterile technique.Aliquot reagent into concentrated batches.Pick solvents in which the reagent is both stable and may readily be dilutedfrom the stock (S) soln to a working soln (W).If the final concentration of the reagent in question is to be compounded in a given solvent, it is often practical to make concentrated stocks of the solvent as well.For example, you wish to investigate the effect of a 3 M ACTH1-39 in Sterno saline. You have a 1.5 mM ACTH1-39 stock (in ddiH20) and a 4x stock Sterno saline soln. You decide you need 1 mL tot of 3 M ACTH1-39 in regular Sterno saline.1st  [3 M/1500 M](1000 L) = 2 L (of the 1.5 mM ACTH1-39 stock)2nd  [1x/4x] (1000 L) = 250 L (4x stock Sterno saline soln)3rd  1000 L tot = 2 L (1.5 mM ACTH1-39 stock) + 250 L (4x stock Sterno saline soln) + X L (ddiH20) 1000 L - 2 L - 250 L = X L (ddiH20) X L (ddiH20) = 748 L (ddiH20)Thus, you combined 2 L hormone stock plus 250 L Saline stock plus 748 L water.3REAGENT ACQUSITION AND STORAGE Concentrations for stock solns reflect the solubility of the compound in a given solvent and integral values of the working soln (e.g., 2x, 10x, 100x, etc.,).This allows for easy serial dilutions.If small volumes are being aliquoted (e.g., 10 L/eppe, 50tot)).Sterile (autoclaved) eppes are carefully removed with gloved hands and their lids quickly closed.Eppes are subsequently labeled with relevant info and placed in a rack.The desired (sterile) solvent is applied to the reagent and it is teased into soln. This soln is mixed in a sterile container (e.g., 0.5, 1.5 or a 2.0 mL eppe, 15 or 50 mL conical tube for larger vol, etc.,).Eppes are opened and filled to the desired vol and closed. Samples are subsequently wrapped with parafilm and placed under the appropriate storage conditions (including containers). QUESTION # 5HOW DO I LABEL MY REAGENTS?To answer this question, you must consider:Reagent total volume.This will determine the size and number of containers necessary to hold each reagent. You MUST put information sufficient such that the contents of the container may readily be determined.Date (this is essential).Compound.Molarity, pH, solvent, volume, etc.,.One may also generate a code for labeled reagents. However, this code must be made available to all who encounter such reagents.You must also be sure that the writing on the reagent container is maintained until the reagent is consumed. This may require;rewriting on the container. the use of alcohol-resistant markers.QUESTION # 6In what order should I approach my experiment?4REAGENT ACQUSITION AND STORAGE 1st  Make all your calculations. Rxn #, conc., # of experiments, # of aliquots for stock and working