Team:Toronto/Experiment-Gel Electrophoresis

• home
• achievements
• team
  • team
  • collaborations
• project
  • description
  • design
  • experiments
  • proof
  • demonstrate
  • results
  • notebook
• parts
  • parts
  • basic_part
  • composite_part
  • part_collection
• safety
• attributions
• human_practices
  • human_practices
  • silver
  • gold
  • impact
  • integrated_practices
• awards
  • software
  • model

Gel Electrophoresis

Introduction

Get started by giving your protocol a name and editing this introduction.

Materials

Procedure

• Part 1: Casting the gel

1. Add 0.5g of Agarose and 50ml of 1X TAE buffer to an Erlenmeyer flask. (Note that TAE buffer in the stock has 50X concentration and needs to be diluted with autoclaved Milli-Q water)

2. Place the flask into the microwave. After this step flask will be hot. Use heat gloves

3. Set the timer to 2 min. Start the microwave oven but stop every 30 sec to swirl the contents of the flask. This helps suspend the undissolved agarose. Continue until agarose particles are dissolved.

4. While hot, add 3ul of 10,000x SYBR Safe into the agarose solution and mix throughly.

5. Set the flask aside for cooling. Occasionally swirl to make the cooling even.

6. Set up the gel tray and make sure that there is no leak by testing it with dH20 and kimwipes.

7. When the flask is cool enough for you to handle easily, pour the solution into the tray.

8. Wait until the gel solidifies.

• Part 2: Loading the gel

9. Make 400mL of 1XTAE buffer per gel (Note that TAE buffer in the stock has 50X concentration and needs to be diluted with autoclaved Milli-Q water).

10. Make a note of what is going into each well and put that in your lab notebook.

11. Determine the DNA concentrations via Nanodrop. (Approximately 100ng DNA per band is required.)

12. Cut a piece of parafilm to use as a mixing surface. Alternatively can use PCR tubes.

13. Mix DNA with loading dye in a 1:1 ratio directly in the PCR tubes using a pipette tip and gently aspirating. (i.e. 10uL of DNA with 2uL of 6x loading dye) Mix by pipetting up and down. Avoid bubbles.

14. Carefully remove the comb of the gel.

15. Place the gel into the gel electrophoresis apparatus with its tray.

16. Add more 1XTAE buffer to cover the gel completely. Buffer should be 1-2mm above the gel. Make sure that all the wells are submerged.

17. Load 10uL of DNA ladder to the first well. Add 10uL of your sample DNA to corresponding wells. (Remember record which wells you load)

18. Put the lid on. The negative (-/black) electrode should be closer to the DNA wells as DNA migrates towards the positive (+/red) electrode. 10.Set the rig to 100V & 60 min. Hit start. 11.Make sure that your DNA does not run off the gel. The loading buffer enables you to track the DNA. 12.The gel apparatus will stop on its own when the time is up. Proceed to imaging the gel. It can sit in the rig overnight if need be.

• References:

• Created by Seray Cicek

• igem wiki:http://local.biochemistry.utoronto.ca/igem/index.php/Analytical_Gel_Electrophoresis

• jove: http://www.jove.com/video/3923/agarose-gel-electrophoresis-for-the-separation-of-dna- fragments

• open wetware: http://openwetware.org/wiki/Agarose_gel_electrophoresis

• lifetechno: https://www.lifetechnologies.com/ca/en/home/life-science/pcr/elevate-pcr-research/agarose-content-with-tips-and-tricks.html

• Syber green catalog: http://www.lifetechnologies.com/order/catalog/product/S33102

• Syber green staining protocols: http://www.lumiprobe.com/protocols/sybr-green-gel-staining

• Changelog

• Transcribed from iGEM 2015 5/26/2016

</div> </html>