REAGENTS, SOLUTIONS AND BUFFERS

A number of complex solutions are used in biotechnology. Prepare solutions

carefully, remembering to check the pH at the appropriate temperature. While
buffer concentrations do not always have to be exact, their pH and type must be
correct, made with the correct acid/base pair. Salt concentrations are usually
critical- small differences in mM or even uM concentrations can ruin experiments.
Solutions are made from the chemicals (solid or liquids) or may be made using
stock solutions. Solutions may be made at Molar solutions, Normal solutions, percent
solutions or even concentrated stocks designated only as 10x, or some concentrated
solution. Some Common solutions: 50x TAE, 10% SDS, TE (10 mM Tris, 1 mM EDTA
, pH 8).
Formulas you will commonly use include the Henderson-Hasselbach equation for
making buffers (pH=pK + log(A/HA)), and to make solutions from stocks, the dilution
equation C1V1=C2V2.
If two or more reagents are in solution, all are put in the same final volume.
Do not make the solutions and mix them together! Some solutions will be more
difficult to get into solution than others, and some are dangerous (exothermic), so
always check the data sheets before making reagents.

MOLAR SOLUTIONS
In order to make a reagent you will need to know the final concentration and volume
desired. It is easiest to set up equations.

For Molar solutions: If using solids, you will need the Molecular or formula weight, and if
using liquids you will need to know its Molar concentration, or its density and MW. You also
need to know the total volume necessary and the concentration you need.
Set up an equation using the number of moles per liter, times the molecular weight, times
the volume needed. The resulting grams are the amount needed to make the proper
solution.

To make 1 L of a 1 M NaCl solution, (MW 58), set up an equation:

1M = 1 mol/L
1 m x 58g = 58g
L m L Add 58g and bring to 1 L.

% SOLUTIONS
You will simply use the formula 1g/100 mL is = to 1% solution.
Always add acid to base. Wear gloves and mask if necessary.
To make a 5% solution of SDS: Add 5 g to 100 mL total volume.

HOW TO MAKE MORE COMPLICATED SOLUTIONS

If a solution has two or more components, you must determine the amount required for
each individual chemical, and then add ddH2O to make the total.

For example, to make a 2M NaCl, 10%SDS solution, you must determine how to make each
component, then put them together with the appropriate ddH2O to make the desired
volume. (Make 1 L)
You will need 116 g NaCl, and 100g SDS, dissolved in water and brought to a final volume of
1L.

HOW TO MAKE BUFFERS

First use the Henderson-Hasselbach equaton to determine the relative ratios needed of
the acid and its conjugate for a given pH. Once you know the relative ratio, determine the
total moles needed for the concentration and volume desired.
For example: You need 1 Liter of 1 M 4.5 pH solution of a buffer HA, which has a pK of 4.5
. Using HH, you determine the ratio of 1:1 for A:HA. Since you need a
1 M solution, you will need to figure out how much of A and HA you need to obtain a total
of 1 m. . In this case it is simple:
x(A) + y(HA) = 1, where x=y. Set up the ratio and solve.
0.5 moles of A and 0.5 M HA is required for this solution. Convert the mol values to g
using the MW, and make the buffer. You can also use stock solutions to make buffers.

HOW TO DILUTE FROM STOCKS

Use the formula C1V1=C2V2
Use C1 as the concentration that you want, V1 as the volume.
C2 is the concentration of the stock solution. Solve for V2, and add ddH2O to bring to the
proper volume.
Our example will be to make 1 Liter of a mixed solution which contains 10mM NaCl and 1%
SDS, using a 1 M NaCL stock and a 20% SDS stock.
Setting up the formulas:
NaCL 1L ( 10mM)= ? (1000mM) ? = 10 mL
SDS 1L (1%) = ? (20%) ? = 50 mL

To make the solution: mix in a smaller volume of ddH2O, add 10 mL NaCl stock and 50 mL SDS
stock, and bring to 1 L