Richard Lab:Agarose Gel Electrophoresis: Difference between revisions
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<center>'''This protocol is for the typical separation and purification of DNA done in the Richard Lab.''' | <center>'''This protocol is for the typical separation and purification of DNA done in the Richard Lab.''' | ||
<br>'''The | <br>'''The [[agarose gel electrophoresis | consensus electrophoresis protocol]] should be consulted if deviating from the procedure outlined here.'''</center> | ||
===Procedure=== | ===Procedure=== |
Revision as of 13:18, 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 ( Mike usually uses 1.3 - 1.4g).
- 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.
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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