Zebrafish Care and Experimental Techniques: Gel Electrophoresis
Materials:
-All materials are EtBr exposed, so wear gloves!
-Materials are kept on a tray on the lab bench
1. TAE solution
2. Agarose
3. EtBr
4. Plate set-up
5. DNA ladder (in fridge)
6. PCR product
For 70mls of 1% gel solution:
1. Add 70 mls of 1X TAE to the flask
2. Add 0.7g of agarose to the flask
3. Microwave the solution for 1:30 mins, watching for bubbles
4. Microwave again until the solution clears and bubbles but does not overflow
5. Allow the solution to cool to 50 degrees celsius (warm to touch)
5. Under the hood, add 3.5uL of EtBr to the solution (before it cools completely!)
5. Stir the solution gently so that the EtBr dissolves
6. Once dissolved, pour the solution onto the gel plate set-up. Make sure the comb is properly oriented and that the screws are tightened.
7. Allow plate to cool for at least 25 mins
8. Place plate into buffer (TAE) and gently remove the comb
9. Add enough TAE to cover the wells
10. Use a DNA ladder, 20 uL in one well
11. Spin PCR tube
12. Add 10uL of DNA loading buffer to a 50uL PCR tube (the buffer should be at least 1/6th of the total solution volume) to dye and weigh down the solution
13. Add PCR solution to a well
14. Connect the voltage channels, make sure the red (+) and black (-) are oriented correctly. Red should be plugged into red and black should be plugged into black. Red should also be on the bottom. (NOTE: when you put the cover on, press hand on clear top otherwise the entire holding device will flip over)
15. Set the voltage to 80 volts for about 30 mins. There should be bubbles rising gently from the bottom to the top of the liquid.
When viewing the gel, make sure to zoom in so you are taking the closest picture possible. You may also need to to manually expose if the auto expose doesn't show bands well.
-All materials are EtBr exposed, so wear gloves!
-Materials are kept on a tray on the lab bench
1. TAE solution
2. Agarose
3. EtBr
4. Plate set-up
5. DNA ladder (in fridge)
6. PCR product
For 70mls of 1% gel solution:
1. Add 70 mls of 1X TAE to the flask
2. Add 0.7g of agarose to the flask
3. Microwave the solution for 1:30 mins, watching for bubbles
4. Microwave again until the solution clears and bubbles but does not overflow
5. Allow the solution to cool to 50 degrees celsius (warm to touch)
5. Under the hood, add 3.5uL of EtBr to the solution (before it cools completely!)
5. Stir the solution gently so that the EtBr dissolves
6. Once dissolved, pour the solution onto the gel plate set-up. Make sure the comb is properly oriented and that the screws are tightened.
7. Allow plate to cool for at least 25 mins
8. Place plate into buffer (TAE) and gently remove the comb
9. Add enough TAE to cover the wells
10. Use a DNA ladder, 20 uL in one well
11. Spin PCR tube
12. Add 10uL of DNA loading buffer to a 50uL PCR tube (the buffer should be at least 1/6th of the total solution volume) to dye and weigh down the solution
13. Add PCR solution to a well
14. Connect the voltage channels, make sure the red (+) and black (-) are oriented correctly. Red should be plugged into red and black should be plugged into black. Red should also be on the bottom. (NOTE: when you put the cover on, press hand on clear top otherwise the entire holding device will flip over)
15. Set the voltage to 80 volts for about 30 mins. There should be bubbles rising gently from the bottom to the top of the liquid.
When viewing the gel, make sure to zoom in so you are taking the closest picture possible. You may also need to to manually expose if the auto expose doesn't show bands well.
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