Molecular Biology Laboratory Topics

General Techniques

Handling Bacteria

Aseptic techniques:

Use a small flame or heater to kill any bacteria on instruments or the mouths of tubes.
Hold tubes on a diagonal angle, since a vertical position will allow contamination from air deposition.

Use bacteria of same age in all experiments, i.e. same generation.

Store "new" bacteria at -70°C. While using bacteria, it may be stored in different ways:

-20°C with 50% glycerol, good up to 6 months
-70°C with 20% glycerol or 7% DMSO, good up to 2 years
Room temperature, sealed plate (with tape) to keep in moisture, a few days (?)

To ship bacteria, dissolve in agar in a small vial. They will last up to a weak, but will grow into several generations.

Bacteria can be grown in an incubator at 35-37°C. Plates should be put upside down to prevent water condensation (colonies will not grow).

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Spin Dialysis

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Gel Electrophoresis

By passing a current through a porous gel that contains DNA at one end, the DNA will migrate towards the positive charge and separate by size. Gels are made of either agarose or polyacrylamide. Agarose gels are used if the segments to be separated are more than 500 bp long. They must be run on TAE buffer (Tris 4 mM, sodium acetate 20 mM and EDTA 1 mM) at pH 7.2. This buffer should be used only once. Polyacrylamide is used to separate segments less than 500 BP long. They must be run in TBE buffer (Tris 89 mM, boric acid 89 mM and EDTA 2 mM). When using gels for DNA sequencing, a very large polyacrylamide gel is needed with a high concentration of urea (5-8 M), using a slightly different buffer (Tris 100 mM, boric acid 100 mM and EDTA 2 mM).

Different concentrations of agarose or polyacrylamide may be used to obtain different degrees of separation between bands. Higher concentrations facilitate separation of small DNA fragments, while low concentrations allow resolution of larger fragments.

Agarose		Polyacrylamide
0.5% (rarely used)	(?)	4% (rarely used)	100 - 1000 BP
0.7% - 0.8%	0.8 - 1.2 kb	6%	100 - 500 BP
1%	0.5 - 1.0 kb	8%	50 - 400 BP
1.5% - 2%	0.2 - 2.0 kb	10%	20 - 100 BP

Note: this table is straight from my notes, but it look funny to me. If it is wrong I apologize.

Separation will also depend on the tridimensional shape of the DNA fragment: supercoiled DNA, being more compact, will travel faster than linear. Linear DNA will travel faster than circular DNA. Gel apparatus may be horizontal or vertical.

The DNA samples are usually run against a marker of known weights or DNA ladder, for example the lambda BstEII digest pictured on the right.

In this example, lane 4 may be from a reaction to restriction-cut a plasmid. If the mixture contains both cut and uncut vector, they will run differently in the gel due to their shape. The upper band is the relaxed uncut (circular) plasmid moving much more slowly. The middle band is the linear (cut) plasmid and the lower faint band is supercoiled uncut plasmid, which can run further in the gel because it is more compact. Lane 5 may be degraded plasmid DNA. Lanes 1 and 2 may be chromosomal DNA, the one in lane 1 being degraded (lane 1 it is supposed to look like a long blob!).

RNA electrophoresis... working...

For better visualization of how to interpret a gel, practice by running a virtual gel in this cool applet from Colorado State University.

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Other stuff

Acidic phenol is made by saturating it with water (or see page 52 step 3).

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Continue to "??" or take a test: [T1] [T2] [T3].

Need more practice? Answer the following review questions:

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