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Team:University College London/Protocols/Electrophoresis
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'''Preparing the Gel''' | '''Preparing the Gel''' | ||
| - | Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water | + | '''Step 1:''' Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water |
| - | Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat. | + | '''Step 2:''' Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat. |
| - | Step 3: Cool solution under running cold water. | + | '''Step 3:''' Cool solution under running cold water. |
| - | Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul) | + | '''Step 4:''' Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul) |
| - | Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles | + | '''Step 5:''' Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles |
'''Running a gel''' | '''Running a gel''' | ||
| - | Step 6: Add 1 part loading buffer to five parts of loading sample | + | '''Step 6:''' Add 1 part loading buffer to five parts of loading sample |
| - | Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm | + | '''Step 7:''' Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm |
| - | Step 8: Add 5ul of DNA ladder to lane 1 | + | '''Step 8:''' Add 5ul of DNA ladder to lane 1 |
| - | Step 9: Add samples to the remaining wells | + | '''Step 9:''' Add samples to the remaining wells |
| - | Step 10: Run at 100 volts for 1hour and 15 minutes | + | '''Step 10:''' Run at 100 volts for 1hour and 15 minutes |
'''Imaging the Gel''' | '''Imaging the Gel''' | ||
| - | Step 11: Place gel in GelDoc 2000 chamber | + | '''Step 11:''' Place gel in GelDoc 2000 chamber |
| - | Step 12: Turn GelDoc 2000 chamber on | + | '''Step 12:''' Turn GelDoc 2000 chamber on |
| - | Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire | + | '''Step 13:''' From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire |
| - | Step 14: Alter the exposure/settings to give a clear image. | + | '''Step 14:''' Alter the exposure/settings to give a clear image. |
TAE - Tris-acetate-EDTA | TAE - Tris-acetate-EDTA | ||
Revision as of 08:45, 1 August 2012
Electrophoresis
Preparing the Gel
Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water
Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.
Step 3: Cool solution under running cold water.
Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)
Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles
Running a gel
Step 6: Add 1 part loading buffer to five parts of loading sample
Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm
Step 8: Add 5ul of DNA ladder to lane 1
Step 9: Add samples to the remaining wells
Step 10: Run at 100 volts for 1hour and 15 minutes
Imaging the Gel
Step 11: Place gel in GelDoc 2000 chamber
Step 12: Turn GelDoc 2000 chamber on
Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire
Step 14: Alter the exposure/settings to give a clear image.
TAE - Tris-acetate-EDTA EDTA - ethylenediamine tetraacetic acid
