215-Lab3A-Pipeting_dilutions - Lab 3A Buffers Pipeting and...

Lab 3A- Buffers, Pipeting and dilutions
3A-1
Lab 3A
Basic Techniques
Demonstration of using a camera.
Expt. #3A-1: Dilute 10X TE Buffer to Make 1X TE Buffer
Expt. #3A-2: Determine the Concentration of an Unknown DNA Sample
Buffers
Many of the experiments performed in molecular biology and biochemistry require
proteins to carry out a particular function, such as binding to DNA or cleaving a substrate. The
activity of these proteins is often very dependent on the pH, salt concentrations, and temperature
of the reaction mixture. In some cases a change of pH from 7.5 to 6.5 or a 10-degree change in
the temperature may cause greater than a 1000-fold reduction in the protein's activity. It is
therefore very important to understand the function of the proteins involved in each experiment
and know their optimal conditions for activity.
As little as 15 years ago, most enzymes used to manipulate DNA were difficult to obtain
because they were purified only by biochemists in research laboratories. In addition, there were
a very limited number of enzymes available. However, as more and more of the techniques in
molecular biology and biochemistry have become commonplace, companies have stepped in and
now provide these enzymes. Many of the techniques that we use in molecular biology and
biochemistry are now provided by these companies in the form of kits that include all enzymes,
reagents, buffers, protocols, and (frequently) controls for the experiment. These kits are often
very helpful, as well as convenient, for carrying out standard procedures. However, it is easy to
get very complacent about just following the instructions and not understanding what is actually
involved at each step of the protocol. You should understand enough about the procedure to
know the function of the kit's buffers, regardless of whether you have to personally make it. A
buffer is not a magic potion but a chemical solution containing a specific mixture of salts,
buffering agents, and sometimes reducing agents, detergents or cofactors, etc., in which each of
the components has a purpose and is included to optimize the reaction.
Solutions
Most of the solutions used in molecular biology and biochemistry are calculated on the
basis of the molarity of the solute. Sometimes a solution will be made on the basis of weight
percent, parts per million (ppm), or normality.
I. Molarity: A solution based on the number of moles of solute in a given volume of solution.
Molarity =
moles of solute
liter of solution

Lab 3A- Buffers, Pipeting and dilutions
3A-2
Example 1: If 2.92 g of NaCl is dissolved in enough water to make 250 ml of solution,
what is molarity of the NaCl? (The molecular weight of NaCl is 58.5 g/mole.)
M =
moles solute 2.92 g
X
1 mole NaCl
= 0.050 moles NaCl
liter of solution 58.5 g NaCl
=
0.050 moles
0.250 liters
= 0.2 M (or 200 mM)
Example 2: Calculate how much NaCl to use to make 300 ml of a 450 mM solution.
A 450 mM solution is 0.45 moles/ liter and 300 ml = 0.3 liter.