SECTION OF BIO-ENGINEERING TECHNOLOGY, UniKL MICET.

LAB 2: GEL ELECTROPHORESIS Introduction Gel electrophoresis is a technique to separate and analyze macromolecus such as DNA and their fragments based on size and charge.

Methodology 1. Assemble the gel casting tray and comb. The comb should not touch the bottom of the tray.

2. Add 0.8 g agarose powder to 80 mL 1.0x TAE buffer. Using a microwave, melt the agarose solution. 3. When the agarose solution has cooled to about 50C (or put the conical flask containing agarose under running tap water so that it is cooled faster), pour solution directly into the casting tray, ensuring that no bubbles get into the gel. (rinse the flask immediately)

4. Allow the gel to cool and it will solidify and become slightly opaque within 20 to 30 minutes (or allow the gel to cool at room temperature for 2 min and put in -20oC freezer for 8 min). Remove black end pieces.

5. Submerge the gel by adding 1.0X TAE running buffer to cover the gel by about a half a centimeter.

7. Carefully remove the comb by lifting it gently at one end, tilting the comb as it comes out. Pulling the comb straight up creates a vacuum in the wells, which tends to lift the whole gel out of the tray. Ensure that the wells are submerged and filled with buffer.

8. Prepare the DNA samples for loading using loading buffer. 9. Load 4 ul of plasmid (or 10 ul of enzyme mix, lab 2; or 5 ul of PCR mix, lab 3) of each sample into individual wells with a gel loading tip.

10. Once all the samples are loaded place the cover on the gel apparatus. Connect the leads so that the black (negative) lead is at the end of the gel containing the wells and the red (positive) lead is at the end of the gel to which the DNA will migrate. Turn on the power supply and set according to the instructor's guidelines. Check the gel after a few minutes. If the dye migrates in the wrong direction, turn off the power supply and switch the leads. Run at a constant voltage of 80 volts. CAUTION: DO NOT REMOVE THE LID FROM THE GEL APPARATUS WITHOUT DISCONNECTING THE POWER LEADS. ELECTRICAL SHOCK MAY RESULT. 11. When the blue tracking dye has migrated about 75% of the distance to the end of the gel (usually within 60-90 minutes), turn off the power supply and disconnects the power leads. Caution, the gel is very slippery!! 12. Carefully transfer the gel into a plastic dish containing staining solution to cover the gel. Set in a dark drawer for 15 minutes. Rinse the gel with tap water. 13. Visualize the DNA with UV light on the Imaging System. 13. Dispose of the gel into the special trash tray. 14. Rinse the light box and tray with water and dry it with paper towels. Questions 1. What is the approximate size of the plasmid that you observed after gel electrophoresis? Why the size is smaller than what is stated in (2)?