Team:British Columbia/Notebook/Protocols/gelverification

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Gel Verification

Supplies:

1. Agerose (1g/100mg for a 1% gel)

2. 1x TBE buffer

3. Gel casting trays, combs and box

4. Loading buffer

5. SYBR® Safe stain

6. Samples

7. DNA ladder: 1 kb ladder and 1kb plus ladder

Steps:

1. To make a 0.8% agerose gel, weight out 0.8g of agarose and transfer into an Erlenmyer flask.

2. Add 100mL of 1x TBE buffer.

3. Microwave till agarose is completely dissolved (be careful to not boil it over).

4. Allow the solution to call down till it can be touched without getting burned.

5. Add SYBR® safe stain (1/10000, eg. 5µL/ 50mL) and swirl to mix.

6. Use tape to tape the side of the casting tray.

7. Pour gel into casting tray and insert comb (make sure there are no air bubbles in the gel.

8. After the gel has solidified remove combs and tape from the casting tray. Fill the gel box with 1x TBE and put the casting tray with the gel in the box so that it is fully covered with TBE.

9. Load samples with 6x dye and 1 ladder into the wells (ca. 12µL each)

10. Run the gel.

• Plug in to power souce, the -ve pole should be plugged in to the side where the sample is loaded and the +ve pole on the other end

• the gel should be run at constant volts; at 110V the gel should be run for about 45min

11. Remove the gel and take a transilluminator image.

12. Cross-reference sample bands with ladder bands to determine their size.