Richard Lab:Agarose Gel Electrophoresis: Difference between revisions
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# Place the gel tray perpendicular in the electrophoresis to create a casting bay. | # 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. | # 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 | # 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. | # 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. | # Swirl the microwaved agarose in the flask until the solution becomes clear. | ||
Line 25: | Line 25: | ||
# Apply 150 volts and run for approximately 60 minutes. | # Apply 150 volts and run for approximately 60 minutes. | ||
# Photograph the gel using the UVP transilluminator system. | # 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. | |||
The amount of agarose to use in your gel depends on the DNA in question. As a rough guide: | The amount of agarose to use in your gel depends on the DNA in question. As a rough guide: | ||
Agarose (g/100mL) DNA resolution (/kb) | <center>Agarose (g/100mL) DNA resolution (/kb) | ||
0.5 1-30 | 0.5 1-30 | ||
0.7 0.8-12 | 0.7 0.8-12 | ||
1.0 0.5-10 | 1.0 0.5-10 | ||
1.2 0.4-7 | 1.2 0.4-7 | ||
1.5 0.2-3 | 1.5 0.2-3</center> | ||
[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]] | [[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]] | ||
Back to [[Richard_Lab | Richard Lab]] | Back to [[Richard_Lab | Richard Lab]] |
Revision as of 12:44, 12 June 2009
Back to Richard Lab
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.
The amount of agarose to use in your gel depends on the DNA in question. As a rough guide:
0.5 1-30 0.7 0.8-12 1.0 0.5-10 1.2 0.4-7
1.5 0.2-3Back to Richard Lab