Distilled water is used to dilute the concentration 20 times to form a 1X PBS solution. Pour the necessary volume of 20X PBS concentrate into a mixing flask and top up with DI water to get the final volume. Stir only briefly. The pH of the 1X solution should be 7.6 +/- 0.2. Check this value using a pH meter or color changes in chemical indicators.
TBS ten times (concentrated Tris-buffered saline) Make a 1x solution by mixing 1 part of the 10x solution with 9 parts distilled water and bringing the pH back to 7.6. The 1x solution has final molar contents of 20 mM Tris and 150 mM NaCl. Store at 4°C.
For some reason, many labs only use tris-hydroxymethyl aminomethane (TRIS) as their Tris buffer instead of using Trizma base. TRIS is acidified with hydrochloric acid to a pH of 7.6 while Trizma base is used unaltered (it's pH is 8.2). Therefore, converting between the two requires adding hydrochloric acid or sodium hydroxide to raise the pH back to 7.6 or 8.2 respectively.
Sodium phosphate buffers are often used in place of Tris buffers. These buffers contain phosphoric acid and sodium hydroxide and have concentrations that are four times greater than a Tris buffer with the same pH. For example, a sodium phosphate buffer at pH 7.0 would contain 200 mM sodium phosphate and 0 mM Tris.
Borate buffers are similar to sodium phosphate buffers but they also contain boric acid or sodium tetraborate decahydrate (10x solution).
2.00 mL of your stock solution should be pipetted into a 500 mL volumetric flask. Add diluent to the flask's mark (you will have added about 498 mL of water). You now have a dilution of your original solution of 1:250. Volumes less than 2.00 mL cannot be accurately diluted so do not attempt this procedure with samples below the threshold of detection by your assay.
As long as you are working with volumes that can be measured with precision and accuracy, you can make any desired concentration by simply repeating this process. For example, if you want to make a 10% solution out of a 100 mg/mL stock solution, just repeat the procedure above using 10% as the volume for the diluent.
When preparing large numbers of samples, it is easier to use a prepared solution of diluent. For example, you can prepare a series of solutions with different concentrations in separate flasks and then combine them after all the samples are ready. This is called a serial dilution and is useful when trying to determine an unknown concentration of substance in the sample.
To produce a 10x buffer, use one component 10x with nine parts water [1+9=10]. 1ml of Z equals 1ml of 10x in 10ml of water. This also means that to produce 1x buffer, add 10-1=9 ml of water to 1 ml of 10x concentration.
For example, if you were making a buffer for the virus plaque assay and the manufacturer suggested a final concentration of 0.5% w/v, then you would need 500 ml of water plus or minus 9 ml to make the buffer 100%.
This calculation is easy to remember because it is based on the formula volume = concentration x total volume which in this case is 500 ml x 0.5% w/v = 25 ml of water. So you would take 25 ml of water and mix it with 1 ml of 10x solution to make your buffer.
Some buffers require different concentrations of salt or other chemicals. But for most buffers, this conversion from 10x to 1x is all you need to know.
1ml of the stock solution (covert 100ug/ml) with 9ml of water yields a 100ug/10ml solution. 1ml of this contains 10 ug/ml. Therefore, the original solution was 100 ug/ml.
A 100ug/ml solution can be made by mixing 100u g of the powder in 1 ml of water and heating it for 15 minutes. Then, let it cool down and store it in the fridge.
The stock solution is stable for 3 months when stored in the freezer.
You can also make a 100ug/ml solution by directly adding 100 ug of the powder to 1 ml of water at room temperature. However, this solution should be used immediately as it tends to precipitate out of the water once it gets cold.
The final concentration of the solution will be 10ug/ml.
Generally, solutions with concentrations lower than 100ug/ml can be prepared by directly weighing out the required amount of the powder and then processing it with some volume of water. But, keep in mind that these solutions are likely to contain less than the expected amount of drug because of possible loss of material during processing.
Solutions containing more than 100ug/ml need further dilution before use.
Pipette out 10 ul of the medication in a test tube using a micropipette to make a concentration of 10 ug/ml. Then dilute it with 990 ul (for a total volume of 1 ml) of the solvent you'll be using in your experiment (ethanol, methanol, water, etc.). You can use any volumetric flask for this purpose.
The solution should be visible by eye under normal light conditions. Use a spectrophotometer to verify that the solution contains no particles or debris and is within 0.1% of its final concentration.
To further concentrate the solution, dialyze it against distilled water until the volume is less than 100 ul. The solution should be stable for at least 24 hours at room temperature, away from light. If not, freeze it in aliquots and thaw only as needed.
The final product should be stored in a refrigerator or freezer.
You can adjust the concentration of your solution by adding more or less drug to begin with. It is important to keep in mind that if you make a too strong solution, it could be harmful to your cell culture wells; if you make a solution that's too weak, you won't be able to see any effects of the drug.
As you may have noticed, this process isn't exactly easy.