Loading Buffers

Gel loading buffers serve three purposes in DNA electrophoresis:
  • Increase the density of the sample: This ensures that the DNA will drop evenly into the well.
  • Add color to the sample: Simplifies loading
  • Add mobility dyes: The dyes migrate in an electric field towards the anode at predictable rates. This enables one to monitor the electrophoretic process

Ficoll based loading buffer

To increase the sharpness of DNA bands, use Ficoll (type 400) polymer as a sinking agent instead of glycerol. The use of the lower molecular weight glycerol in the loading buffer allows DNA to stream up the sides of the well before electrophoresis has begun and can result in a U-shaped band. In TBE gels, glycerol also interacts with borate which can alter the local pH.

This information taken from the following website:


Ingredients used in Microbiological Media

The Manufacture and Purpose of These Ingredients
(All formulas refer to one liter. Thus "10 grams of agar" means 10 grams per liter.)
The early microbiologists used the foods available for homes and restaurants as foods for their bacteria. They supplemented these with blood, fruit juices and other materials found in the environment where they found the bacteria, yeasts, and fungi they wished to study. When viruses were discovered, living cells became "media".
Any microbiological medium and environment must provide everything the species under cultivation requires. These are oxygen (or other electron acceptor), water, nitrogen source, carbon source, energy source, minerals, vitamins, and trace biochemicals. Many bacteria can use glucose as energy and carbon sources. Some bacteria can use light as energy source and others can oxidize sulfur as energy source. As nitrogen source, most bacteria require protein, peptides, or amino acids, but many can use ammonia, nitrates or nitrogen molecules. Bacteria capable of fermentation can oxidize one molecule and use another as electron acceptor; thus, they are not able to obtain as much energy as would be available were the energy oxidized completely to carbon dioxide and water.
Some organisms can make almost everything them need, but others require a long list of vitamins and required factors. Some metabolize their food down to carbon dioxide and water, others have very limited in disimilatory metabolism and leave their food practically untouched. Bacteria used in food processing typically are able to use only a few of the components of the food they used to process. Knowledge of what a bacterium needs helps you design a medium for growing it.


All bacteria require some amount of water. Some can live in evaporating salt water or moist pastes, but completely dry foods such as a bag of sugar are free from bacterial attack. Dried meat or fruit are free from attack by most bacteria, but those foods may be attacked when the humidity increases. However, attack of damp dried foods is usually by fungi rather than bacteria.

Advanced experiments may require distilled water when you are testing exactly which minerals are required by an organism. When growing protozoa or animal cells, the chlorine in tap water may kill the animals.


Agar is used to gel bacteriological broth (liquid media) to form slants, and to avoid sloshing so oxygen can't easily get to the bottom of the media. Agar is obtained from kelp. There are many different grades of agar and most were developed for use in human food. The amount of agar needed to gel broth varies but 10 grams per liter is commonly used. For a harder drier agar, use 12 or 15 grams and for special purposes 20 or more grams is used. For top agar and motility assays use about 7 grams of agar per liter.
There are several kinds of agar which are long chain polymers made by certain marine alga. Commercial agar is often a mixture of molecules having some what differing chemical structures. Agar from different algae have different structures, and, therefore, have differing properties. While some marine bacteria can use agar as food and form pits when spread on a plate of agar, most bacteria can't damage agar.
Since agar contains many different molecules and impurities, some companies fractionate agar and sell special products for use in foods and laboratories. Alginic acid is used in ice cream as a smoothener. In the presence of calcium ions alginic acid forms gels. Therefore, if you mix bacteria with alginic acid and drip the suspension into calcium ion solution, you obtain soft spheres containing trapped bacteria. You can then pass food thru the bed of spheres and food would diffuse into the spheres and the products will difusse out. If the soft sphere pack tightly, you can pass the flow upward in the "fluidized bed".

Agarose is another product obtained by fractionation of crude agar. Agarose is used to form special quality gels for electrophoresis of large molecules such as DNA. Ordinary agar and chemical gels have networks of polymer threads too dense to permit passage of the huge DNA molecules.

Noble Agar is obtained by cutting an agar gel into cubes and passing distilled water upward through the tiny cubes to wash out salts and other small molecules which diffuse out of the agar.

Manufacture of Agar
Agar comes from huge alga plants known as kelps. These are harvested from the sea by floating devices or ships. The kelps are cooked with dilute acid and filtered, the acidic liquid is neutralized and cooled so that an agar gel is obtained. This is cut into blocks and frozen. When the blocks are thawed the agar is in shreds and the water drains away. In the old days, these dried shreds were sold as agar-agar and used in cooking. Professor got the idea of using gelatin gelled media as a solid for bacteriological work, but it did not work very well because when incubated at moderate temperatures, the gelatin melted. Frau working in his lab knew about agar-agar used in cooking and suggested he try using agar.

Carbon Sources

Glucose is the ideal carbon source for many bacteria. Many can make everything they need from glucose and a nitrogen source. Some bacteria can't use sugars and need carbon dioxide, carbonate or bicarbonate. Bacteria found in the cow's rumen do better on organic acids because glucose is rare in that environment and they have developed mechanisms to fit the environment.

Energy Sources

Most bacteria, yeasts, and fungi do best when glucose is provided as the primary energy source because they may not be able to digest other carbohydrates. All bacteria found on and in animals and most of those found on plants and soils do well on glucose as the sole carbon and energy source. However, bacteria which use light or oxidation of minerals for their energy source may do better in relatively organic-free media. In water we find many little studied bacteria which do better in media containing only 0.5 gram of peptone and 0.5 gram of starch.

Nitrogen Sources

Bacteria require nitrogen to make proteins and nucleic acids. Only a few genera of bacteria can use free molecular nitrogen from the air. Others require fixed nitrogen in the organic or inorganic form. Some can use nitrate or nitrite salts, but most require amino acids, peptides, peptones, or proteins.

Peptones are the most widely used source of nitrogen in microbial media. Some are made by cooking milk or meat products in acid, but most are made by incubating milk or meat with trypsin, pepsin, or other proteolytic enzymes to digest the protein to a mixture of amino acids, peptides, and polypeptides. Many microbes, called proteolytic, can digest proteins, but most can't. The choice of peptone is sometimes of importance. For example, for a positive hydrogen sulfide test, the medium must contain sulfur-containing amino acids.

Tryptone is a tryptic digest of casein. Casein is a complex of proteins found in milk. Trypsin is an important digestive enzyme produced by the pancreas. It cleaves proteins into shorter pieces called peptones. Tryptic digests of dried milk are called tryptones. Tryptones are the best choice for bacteria media because they are used by most bacteria from animals and supply nitrogen, energy, and carbon. Tryptone water (tryptone + water) will support the growth of many species of bacteria. Tryptones are not pure substances. They are a mixture of left over trypsin, salts, and vitamins, amino acids, peptides, peptones (longer than peptides), and polypeptides (longer than peptones). Tryptones are used in foods for flavoring and nutrition, in electroplating for smooth plating, and in media for microbes.

Not all tryptones are identical. Many manufactuers make several grades of tryptones. Some dissolve in water to give a totally transparent yellowish solution free of any turbity or sediment. These expensive tryptones are often used in diagnostic media when super clarity is needed. Some tryptones are much cheaper and form quite turbid, darker colored solutions with some sediment and often support better growth of microbes. These are commonly used in manufacteuring antibiotics and other drugs.
Phytone is a tryptic digest of soybean proteins. Some bacteria isolated from plants may grow a little better on phytones than on tryptones.

Peptones are enzymic digests of other proteins often meat scraps.



Nitrates as honorary oxygen.


Yeast Extract

Yeast extract is used in most media for microbes from plants and animals. Some of these microbes require vitamins and other growth factors from their plant or animal hosts and yeast extract is rich in vitamins, minerals, and digested nucleic acids. However, yeast extract is very hydroscopic and is difficult to keep dry in the classroom and is sometimes difficult for students to obtain. See YIB on page b030 for a method for making your own yeast extract.

Commercial preparation of yeast extract begins with a tanker load of waste yeast from a brewery. The yeast is dumped to huge stainless steel tanks and it is incubated about 50C. At that temperature, health of the cell is ruined and the internal partions degrade and enzymes begin at the cell from within (autolysis = self lysis). This process of self-lysis occurs in all cells at death. After a day or so, stirring stops, and the liquid is separated from the solids (yeast cell walls and bits of hops and grain). The wonderfully flavored yeast extract is then used for icecream syrup, candy, breakfast foods, gravy to add flavor and vitamins, sugars, and nucleic acids and other nutrients. A tiny part of the production is used in bacterial medium to replace some or all of the meat extract. Yeast extract is high in B-complex vitamins.

It is easy to make. If neccesary you can grow your own yeast, but you can usually buy compressed yeast at any bakery. I usually comes in one pound foil wrapped bricks.

YIB is excellent for cultivation of lactic acid bacteria. For even better growth of the bacteria, do not remove the spent baker's yeast. Filter and use egg only if needed for clarity.

Notes on Choice of Ingredients

Scientists use high quality distilled water when doing exacting nutrition work, but tap water gives good results in the classroom. However, tapwater may give a cloudy preciptate in some instances. I use spring water, but tapwater is just as good. If it has lots of chlorine or junk, you may let it stand a few days before use--especially if you are growing animals in the water.

MgSO4.7 H2O is Epson Salt.

NaCl is table salt, but use normal salt not Morton's which contains insoluble aluminum salts which should be harmless but will give cloudy medium. I urge you to use only salt that dissolves to a clear solution. Afterall, aluminum is the active ingredient in underarm deodorants.

If you don't have all the minerals such as Mg, Ca, Mn, etc. stir soil into spring (or tap) water and let the mud settle a couple days and use the clear supernatant water.

If the chemical you have differs in water of hydrate. Solve a proportion to get the correct amount or just ignore the difference. You can't ignore the difference if you are preparing pH buffers by weight.

Calculating PCR Cocktails

How to calculate primer concentration?
Example 1: the COA specifies we have 24 nmole of oligo. If we resuspended oligo in 1 ml:

1 ml = .001 L
24 nmole/0.001 L = 24000 nmole/L or 24000 nM
24000 nmole/L X 1 umole/1000 nmole = 24 umole/L or 24 uM
Explanation: We convert the volume in which the oligo was resuspended into liters. Then the total nmole amount of oligo is divided by the volume to get nM conc. The nmoles are converted to umole to get the uM conc.

Example 2: making 100 uM primer stock:

If COA specifies we have 24 nmole of oligo:
24 nmole X 1umole/1000nmole = 0.024 umole
0.024 umole/100 umole/liter = 0.00024 L
0.00024 L X 1000 ml/L = 0.24 ml or 240 ul
Explanation: We convert from nmole to umole then divide by the desired concentration in umole/L. The umoles cancel out giving the needed volume in liters. We then convert liters to ml. So in this example the oligo should be suspended in 0.24 ml to get a 100 uM solution.

Example 3: Calculate from OD. If primer OD is 0.14:

If the ug/OD reported on the COA is 36.6:
0.14 OD/ml X 1000ul/10ul = 14 OD/ml stock
14 OD/ml X 36.6 ug/OD = 512.4 ug/ml
Explanation: The OD/ml is multiplied by the dilution factor to get the stock OD/ml. The OD is converted to ug/ml by multiplying the OD/ml of the stock by the ug/OD conversion factor listed on the COA. The ug cancel out giving ug/ml.

If the nmole/OD reported on the COA 4.9:
0.14 OD/ml X 1000ul/10ul = 14 OD/ml stock
14 OD/ml X 4.9 nmole/OD = 68.6 nmole/ml
68.6 nmole/ml X 1000 ml/L = 68,600 nmole/L or 68,600 nM
68,600 nmole/L X 1 umole/1000 nmole = 68.6 umole/L or 68.6 uM
Explanation: The OD/ml is multiplied by the dilution factor to get the stock OD/ml. The OD is converted to nmole using the conversion factor on the COA. Then ml are converted to liters and nmole are converted to umole to get the uM concentration.

Example 4: Calculate from MW. the COA specifies a molecular weight for the oligo of 7440.0:

7440.9 g/mole = 7440.9 ug/umole
7440.9 ug/umole X 68.6 umole/L = 510445.74 ug/L
510445.74 ug/L X 1 L/ 1000 ml = 510.4 ug/ml
Explanation: g/mole is the same as ug/umole. The molecular weight expressed in ug/umole is multiplied by the uM concentration determined in example 3. The umole cancel out leaving ug/L. Liters are converted to ml to give the ug/ml concentration.
What is the recommended concentration of magnesium chloride in PCR?
The MgCl2 should be optimized for each template and primer pair. In general the final concentration varies between 0.5 and 2.5 mM (when using 0.2 mM dNTPs). EDTA or excess dNTPs can inhibit amplification by chelating the magnesium ions necessary for Taq DNA polymerase activity.
How do I determine the appropriate annealing temperature?
The annealing temperature should be 5°C below the melting temperature (Tm) of the amplification primers. Temperatures in the range of 55°C to 72°C generally yield the best results. The Tm can be determined by most computer programs used to design primers.
The general formula Tm = 4(G+C) + 2(A+T) may be used to determine Tm of oligonucleotides less than 10 bases in length. For oligonucleotides greater than 10 bases, use the following formula:[67.5 + .34(%GC as a whole number) - 395/length of the oligonucleotide]