Richard Lab:Agarose Gel Electrophoresis: Difference between revisions
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{| border="1" | {| border="1" | ||
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! Agarose (g/100mL) !! | ! Agarose Concentration(g/100mL) !! Optimal DNA Resolution (kb) | ||
|- | |- | ||
| 0.5 || 1 - 30 | | 0.5 || 1 - 30 | ||
|- | |- | ||
| 0.7 | | 0.7 || 0.8 - 12 | ||
|0.8 - 12 | |- | ||
| 1.0 || 0.5 - 10 | |||
|- | |||
| 1.2 || 0.4 - 7 | |||
|- | |||
| 1.5 || 0.2 - 3 | |||
|} | |} | ||
Revision as of 12:56, 12 June 2009
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The consensus electrophoresis protocol should be consulted if deviating from the procedure outlined here.
Procedure
- Place the gel tray perpendicular in the electrophoresis to create a casting bay.
- Wipe the tray clean with a Kim-wipe and level the tray using the bubble level.
- Weigh out the desired amount of agarose and add it to 100ml of 1X TBE in a 300ml flask.
- Microwave the flask on high for 80 seconds. Be sure that it does not boil over.
- Swirl the microwaved agarose in the flask until the solution becomes clear.
- Pour the melted solution into the casting bay and insert the comb.
- Prepare the DNA ladder by combining the following:
- 10ul DNA ladder
- 1ul SYBR green (100X)
- 1ul Bromophenol Blue
- Prepare the DNA Samples by combining the following:
- 40ul DNA preparation
- 4ul SYBR Green (100X)
- 4ul Bromophenol Blue
- Remove the comb from the cured gel and realign the gel in the chamber.
- Adjust the buffer level by adding 1X TBE to the chamber until the buffer just covers the gel. The buffer can be reused a few times.
- Pipette the samples into the wells
- Apply 150 volts and run for approximately 60 minutes.
- Photograph the gel using the UVP transilluminator system.
Notes
- Our "1X TBE" is technically 0.5X TBE but for our purposes we call it 1X TBE.
- Correspondingly, the 10X TBE is officially 5X
- Use the following table to determine the amount of agarose you want to use.
0.5 1-30
0.7 0.8-12
1.0 0.5-10
1.2 0.4-7
1.5 0.2-3
Agarose Concentration(g/100mL) | Optimal DNA Resolution (kb) |
---|---|
0.5 | 1 - 30 |
0.7 | 0.8 - 12 |
1.0 | 0.5 - 10 |
1.2 | 0.4 - 7 |
1.5 | 0.2 - 3 |
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