Electrophoresis

Electrophoresis

I. Purpose

· perform electrophoresis using restriction enzymes and lambda DNA

· understand how a restriction enzyme works

· analyze a photograph of electrophoresis

· understand how gel electrophoresis separates DNA molecules in a mixture

· how to use electrophoresis to separate DNA fragments

· determine unknown DNA fragment sizes when given DNA fragments of known size

II. Materials

agarose powder

casting tray and comb

camera

crushed ice container

distilled water

DNA samples

electronic scale with tare

electrophoresis box

250 ml Erlenmeyer flasks

film

graduated cylinder

hood

ice

loading dye

microcentrifuge

micropipet and tips

1.5 ml reaction tubes and racks

restriction buffer

restriction enzymes (HindIII, EcoRI, BamHI)

10x TEA buffer

UV filter

UV transilluminator

37º C water bath

weighing boat

III. Procedure

Place the weighing boat on the scale and tare. Weigh out 0.8 ml of agarose powder and place it into a 250 ml Erlenmeyer flask.

Add 10 ml of 10x TEA buffer and 90 ml of distilled water into a graduated cylinder to create a 1x TEA buffer solution. Add this to the Erlenmeyer flask containing the 0.8 ml of agarose.

Dissolve and boil the agarose solution in a microwave, about 2-3 minutes.

Place clean bottom of the casting tray in place, and pour in the agarose solution. Place the casting comb in place. Allow gel mold to set undisturbed until almost opaque, about 10 minutes.

Fill a graduated cylinder with 50 ml of 10x TEA buffer and 450 ml of distilled water, creating 500 ml of 1x TEA buffer.

In each of the four restriction enzyme tubes, combine 1.0 µl of restriction buffer, 7.0 µl of distilled water, 1.0 µl of the specific enzyme (eith...

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...tion enzymes are found naturally in bacteria. They act as a protection against viral infections, as they break down incoming viral DNA.

4. The electricity in electrophoresis acts on DNA as a magnet does to another magnet. DNA has a slightly negative charge. The samples containing DNA are loaded at the cathode or negative end. When the power is activated, the DNA is attracted towards the positive end of the electrophoresis box. The agarose gel provides a means of slowing the DNA down. The DNA fragments must work through the gel matrix in order to reach the end.

5.

6. The loading acts as a point of reference. It allows the person performing the experiment to see how far down the DNA sample has moved. The DNA is photographed using ethidium bromide, a UV-sensitive substance and an ultraviolet transilluminator to highlight the DNA strands.