Biotechnology and Bioinformatics (1+1)

A. Electrophoresis of a DNA sample of unknown concentration with a known standard

1. Place the gel plate into gel mould, position the comb and ensure that the gel is horizontal – check with a spirit level is necessary (different supplier have different designs, do follow the instructions from manufacturers).

2. Prepare a 1% agarose gel: dissolve 1g agarose in 100 ml 0.5x TBE or 1x TAE. Heat the mixture in a microwave oven until completely dissolved.

3. Cool to 60°C.

4. Pour agarose onto the gel tray and allow it to set for at least 30 min.

5. Remove the comb. Place the gel into the electrophoresis tank and pour 0.5xTBE or 1xTAE (same as the buffer that was used to make gel) until the gel is completely covered.

6. Mix 1 μl loading dye and 2 μl DNA and load into the well.

7. Load 2 of DNA marker (Hind III digested λ DNA for example) into one of the wells.

8. Run the gel at 70-100V until the dye is about 2.5 cm from the origin.

9. Move the gel to a tray with ethidium bromide (1μl ethidium bromide in 100 μl ddH₂0) (HAZARD!!!).

10. Let the gel stain for 5- 10 min, and then de-stain for about 2 min in another container with ddH₂O only.

Illuminate the gel with UV light (CAUTION – UV LIGHT IS HAZARDOUS!!! – WEAR MASK OR UV PROTECTION GLASSES IF EXPOSED TO UV LIGHT).

11. Photograph the gel under the UV.

12. Compare the intensity of the DNA bands of the samples with the intensity of the λ bands. As the amount of DNA present in each λ band is known (information is often provided by the supplier), the amount of DNA of each sample can be estimated by comparing the fluorescent yield of the sample with those of the λ bands.

13. Quality of extracted DNA can also be assessed by looking at the gel. Good quality DNA will show as a sharp intense band. Degraded DNA extracts will show various degree of smearing.