Team:Debrecen-Hungary/protocols/Gel electrophoresis

Gel Electrophoresis:

Scientific Background

Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

Overview

Materials

agarose

sterilized bottle

1x TAE

microwave

G-red

ladder

pippett(0.5-10µl)

Gel electrophoresis apparatus

Procedure

1. masure 1 g agarose (for 1% gel agarose).

2. put it into a sterilized bottle.

3. masure 100 ml 1x TAE.

4. put the bottle into the microwave (must not close the bottle totally), heat it still it will be fully clear.

5. Take the bottle out from the microwave with the plastic gripping.

6. Cold down the bottle in water bath until you can touch it.

7. Add 100 µl G-red into the bottle (because 1000x must attenuate).

8. Shake gently the bottle still the red color disappears.

9. Put the liquid from the bottle into the gel tray (if there are any bubbles in ti you can punch them with a tip)

10. After it became solid turn the gel rack to 90°.

11. Put 1% TAE into the gel tray still the level of line on the wall

12. Take out the comb andclean it

13. Put a ladder (3-6 µl) into the first hole

14. Into a new tube mix the loading dye(1 µl) and your sample(5 µl)

Notes & troubleshooting

References

1. Theriogenology. 2010 Sep 10 ,Huang HW, Su YF, Yao CT, Hung YC, Chen CC, Cheng CC, Li SS, Chang HW. "High-throughput gender identification of three Columbidae species using melting curve analysis"

Links

[http://www.youtube.com/user/debrecenigem2010#p/u/22/Q-kmMidAo3A Video I]

[http://www.youtube.com/user/debrecenigem2010#p/u/21/hjKJzMoa4_g Video II]

[http://www.youtube.com/user/debrecenigem2010#p/u/20/h6fVSlELFbo Video III]