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Dilutions Preparing a more concentrated solution which will be diluted: Question: You know that your total volume will be 120 ul. You want a [final EGC]=50 ug/ml. You know that you will be added 30 ul of EGC. At what [stock] do u need to prepare. Answer: The dilution factor here is 4 since 120/30=4. so take your 50 x 4= 200 ug/ml. That is the concentration you should prepare. Alternative way: V1xC1=V2xC2 30ul x C1 = 120 ul x 50 ug/ml C1=200ug/ml To prepare for a finale [100 ug/ml] you would simply multiple this by 2 or 400 ug/ml q. how would u prepare this 400 ug/ml stock if your starting stock is 1000 ug/ml? V1(1000 ug/ml) = 1ml(400 ug/ml) V1=.4 ml = 400 ul if you wanted only 0.5 for Volume 2 would take out 200 ul of this Question: Your bottle of Dextran-FITC is a solid at a concentration of 100 mg/ml. You would eventually like to have a final concentration in your test tube of 3 ml of 1mg/ml. How should you prepare your stock and dispense? If we were to add 1 ml of H2O, the concentration would be 100mg/ml adding 2 ml of H2O would halve this cocentration to 50 mg/ml and adding 4 ml would make it 25 mg/ml. Lets work with 25 mg/ml V1xC1=V2xC2 25,000 ug/ml V1 = 1000 ug/ V1= .012 ml or 120 ul Rather then using all of your solid in the stock tube, it may be more convenient to weight out a portion of this and prepare a stock concentration from that. Lets try weighing out 25 mg. To do this weight out .025 g. When you add 1 ml to this you will have 25 mg/ml or 25,000 ug/ml. The calculations would then be the same as above. Question: This time your Bottle of Dextran comes at 1g. Again, you would like to use a final concentration in your cell cultures of 1 mg/ml. If you were to add 1ml, you would have 1 g/ml or 1000 mg/ml or 1,000,000 ug/ml. If you were to work with this V1xC1=V2xC2 1,000,000 ug/ml V1 = 1000 ug/ V1=.001 ml = 1 ul (This is a very small amount to add. so it would be better to work with a more diluted stock. If you were to add 4 ml, you would have 250 mg/ml or 250,000 ug/ml. But if you do the calculations above you would still add only 4 ul. Another way you could deal with this is to weigh out some of the powder stock. How about if you way out 20 mg? On the scale 20 mg is 20/1000 or .020 g which is equal to .020 g or 20,000 ug. V1xC1=V2xC2 20,000 ug/ml V1 = 1000 ug/ml (1ml) V1 = .05 ml or 50 ul. So you could weigh out .020 grams and add 1 ml to this tube. From this working stock you could then add 50 ul to your 1 ml tubes. Question (diluting PBS): Your stock PBS comes at 5x and you want to make up 500 ml of 1x. You should add 100 ml of your 5x PBS to 400 ml of H2O. Question (diluting sulfuric acid): Your bottle of sulfuric acid comes at 36N and you need a 1N solution. You would like to make 100 ml. How much water and how much sulfuric acid should you add? (36N)(X)=1N(100ml) X=2.7 ml So you could add 97.3 ml of H2O (always add water before acid) and then 2.7 ml of acid. To make this even another thing you could do would be to add 3 ml (36N)(3)=1N(X) and 108 ml H2O since X=108 Question: going from a more concentrated solution to a less concentrated one:: Your protein concentration is 4580 ug/ml. You would like to take 5 and 10 ug from this stock and place into your wells. (1) If you dilute to 500 ug/ml, you will need to take 20 ul of this to get 10 ug/ml. (500ug/ml)V1=(10 ug/ml)(1ml) =.02ml or 20 ul To get this from your stock in 1 ml dilution buffer: (4580ug/ml)V1=(500 ug/ml)(1ml) V1=.109 ml or 109 ul So add 109 ul of your stock to 891 ul of your buffer. From this you are now ready to add 20 ul for you 10 ug/ml (2) Now do a 2 fold dilution from (1) which gives you 250 ug/ml and you would need to take 5 ul out of this to get 5ug/ml. (250ug/ml)V1=(5ug/ml)(1ml) V1=.02ml or 20 ul So take .5 ml from (1) and add to .5 ml for your 2 fold dilution. Question (Dilutions/secondary antibody): Prepare a secondary antibody diluted 1:10,000 in dilution buffer. 1 ul ------10,000 ul 1 ul ------10 ml (this would make a nice easy dilution) Question (Dilutions/secondary antibody): Prepare a secondary antibody diluted 1:1000 in dilution buffer. 1 ul ------1ml Question: Prepare a 1,000x stock solution of BSA having a concentration of 100 ug/ml BSA. A 1,000x concentration would be 0.1g / 1 ml or 1g/10ml. So add 1g BSA for every 10 ml PBS buffer (100
Question: You have prepared 15 ml of a 1000x concentration stock solution as above and now want 15 ml 1x concentration of the same solution. How do you dilute the solution? 1ml --per--1000ml = 1x 1ul --per-- 1ml = 1x 15 ul --per-- 15ml = 1x (this is want you want. so add 15 ul of the concentration solution for every 15 ml buffer) Question: You have 80 ml media and want a final [GMSF]=10 ng/ml. You have a vial with a drop in it marked 200 ng. How should you prepare? If you add 1 ml to the vial, the concentration will be 200 ng/ml. You will need to dilute this to get your [desired]. 200 ng in 1 ml = 200 ng/ml 200 ng in 10 ml= 20 ng/ml (take the 1 ml and place it in 9 ml media) 200 ng in 20 ml = 10 ng/ml (take the 1 ml and place in 19 ml media) so you can just take the 1 ml, transfer it to a 2nd via, then to a 3rd and then to a 4th and then add this to 79 ml of media. Question (Diluting LPS): You would like to have 3 concentrations of LPS; 1 ug/ml, 100 ng/ml and 10 ng/ml. Your starting concentration of LPS in the vial is 1000 ug/ml. Dilution 1 (to prepare 100 ug/ml): This is 1/10 of 1000. So do a 1:10 dilution. You could do this by say take 20 ul of the LPS and placing it into 180 ul of (anything, pbs, etc). Dilution 2 (to prepare 10 ug/ml): This is 1/10 of Dilution 1. So can take out 20 ul from Dilution #1 and place into 180 ul anything. Dilution 3 (to prepare 1 ug/ml): This is 1/10 of Dilution 2. So can take 20 ul out of Dilution 2 and add 180 ul PBS. To prepare 100 ng/ml: One way is to take 10 ul out of dilution #2 and place it into 1 ml because 10 ug/ml = 10,000 ng/ml So 10,000 ng/ml V1 = 100
ng/ V1 = 10 ul To prepare 10 ng/ml: Might be easier to work with Dilution 3. Would need to take 10 ul out of that dilution because 1 ug/ml = 1000 ng/ml Otherwise will be taking only 1 ul out of Dilution 2 So 1000 ng/ml V1 = 10 ng/ V1= 10 ul Question (multi-well plate): You know that your finale volume in your multi well should be 200 ul. You want final concentrations of 10, 50, 100, 150 in your wells. Your [EGCG] stock is 5000 ug/ml. How should you add this? If you were to try to do this direction for the 10 ug/ml concentration you would end up added .4 ul which is impossible to measure and add. So it would be wise here to dilute your concentrated stock. Dilution 1 (to prepare 1000 ug/ml) stock: This is 1/5 of 5000. So do a 1:50 dilution. Could either do 20 into 80 ul or 40 ul into 160 ul of anything. Now your can prepare your 10 ug/ml concentration. 1000 ug/ml V1 = 10 ug/ml (.2ml) V1=2 ul if you want could dilute 1 even further and work with that For the 50 ug/ml would add 10 ul For the 100 ug/ml would add 20 ul and for the 150 would add 30 ul Dilution 2 (to prepare 100 ug/ml) stock: Since by adding 1 ml to the EGCG tube will have this concentration could use this dilution to more easily achieve the EGCG 10 and 50 concentrations. 100 ug/mlV1 = 10 ug/ml (.2ml) V1=20 ul For the 50 ug/ml would add 100 ul. Question (multi-well plate; alternate): As an alternate way to the above, you could also prepare your cells and dilution all in separate tubes and then dispense this into multi well plates. To do this, prepare 2x the concentration of EGCG which you need. Also prepare 2x concentration of the cells you need. When you add the appropriate amount of cells and EGCG you will obtain the correct concentration which you can dispense into your wells. 1stTo prepare 150 ug/ml EGCG Take 30 ul of your 5 mg/ml stock into 470 medium 5000 ug/ml V1 = 300 ug/ml (500 ul) V1=30 ul To prepare 100 ug/ml EGCG Take 30 ul of your 5 mg/ml stock into 470 medium 5000 ug/ml V1 = 200 ug/ml (500 ul) V1=20 ul To prepare 50 ug/ml EGCG Take 10 ul of your 5 mg/ml stock into 490 medium 5000 ug/ml V1 = 100 ug/ml (500 ul) V1=10 ul To prepare 10 ug/ml EGCG Take 2 ul of your 5 mg/ml stock into 498 medium 5000 ug/ml V1 = 20 ug/ml (500 ul) V1=2 ul 2nd Bring your cells up 2x. So if want [cell] at 1x106 you would add 1/2 the amount of your cell # (ex. you have 5.5x106 cells, so bring up into 2.75 ml rather then 5.5. 3rd Now add 500 ul of your cells to each of your concentrations above. You now have your cells and EGCG at the correct concentration. Dispense 200 ul of each tube into each well. (you would also need to dispense 100 ul of cells at 2x + 100 ul media if you want cells only wells.
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