Electrophoresis Essay Research Paper I Purpose perform

Electrophoresis Essay, Research Paper

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

37? 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 ?l of restriction buffer, 7.0 ?l of distilled water, 1.0 ?l of the specific enzyme (either HindIII, EcoRI, or BamHI). For the control, add no enzyme. Close the caps tightly and place them evenly balanced in the microcentrifuge and spin for 2-3 seconds. Place the tubes in the 37? C water bath.

When the gel has solidified remover the comb in a careful straight up motion. Remove the glass plate bottom without disturbing the gel and place it in the electrophoresis box with the wells towards the cathode end. Pour the prepared 1X TEA buffer carefully over the gel until the liquid level completely covers the gel and is about 1 or 2 mm above the surface of the gel.

Add 2 ?l of loading dye to each of the enzyme tubes using the micropipet and spin them in the centrifuge. Extract 10 ?l of the first sample and load it into the first well. Repeat this with the other samples, changing tips between each.

Attach the power supply to the electrophoresis box. Set it to 100 volts and 40 milliamps and activate it. After about 45 minutes or until the dye is approximately ? of the down, turn off the power supply and disconnect the box. Using gloves, remove the gels from the box and place them on the transilluminator.

The instructor will carry out the photography of the electrophoresis gel.

Clean the lab area.

IV. Observations and Results

HindIII EcoRI BamHI Control

Distance Act. BP Distance Cal. BP Distance Cal. BP Distance Cal. BP

3.4 cm 25,000* 3.5 cm 23,000 3.8 cm 19,000 3.7 cm 20,000

4.8 9,416 5.3 7,800 4.2 15,000 ———— ————

5.9 6,557 6.4 5,200 5.7 6,800 ———— ————

6.7 4,361 7.1 4,000 5.9 6,500 ———— ————

11.3 2,322 8.7 3,300 6.7 4,300 ———— ————

12.1 2,027 ———– ———— ———— ———— ———— ————

* = rounded base pair

All calculated base pairs (Cal. BP) are rounded figures.

V. Conclusions

Electrophoresis literally means “to carry with electricity.” It is the use of restriction enzymes and electrical current to measure segments of DNA from a sample.

Restriction enzymes are enzymes found in bacteria. These are enzymes that are able to cut through the phosphate-sugar backbone of DNA at restriction sites. Restriction sites are certain base sequences recognized by these enzymes. In bacteria, restriction enzymes act as a defense against invading viruses. When the viral DNA is release into the cell, the restriction enzymes cut it into pieces, rendering it useless and unable to act upon the cell. Any other bacteria entering the cell will also be cut if it contains the base sequence recognized by the enzyme. Every species of bacteria has at least one restriction enzyme. Restriction enzymes are used in genetic engineering to make complementary cuts that allow the insertion of a genetic code into a genome. In electrophoresis, restriction enzymes cut at the restriction sites on the DNA sample. It cuts as many times as the base sequence appears on the sample.

After the sample is cut, buffers, dye, and a substance called ethidium bromide is added to the sample. It is then placed into the well of an agarose gel. An electrical current is run through this, and because DNA has a negative charge it is dragged through it towards the positive end. The DNA weaves through the agarose gel, the smallest pairs going the farthest simply because they are more maneuverable. The longer segments move

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