Magni:Protocols/tecan: Difference between revisions
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(New page: {{Magni}} <div style="padding: 10px; width: 720px; border: 5px solid #000000;"> = Infinite F200 readings = The microplate reader can be used for static or dynamic readings of bacteria cul...) |
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=== Static readings === | === Static readings === | ||
* Grow bacteria under the desired conditions. | * Grow bacteria under the desired conditions. | ||
* Transfer 200 ul of | * Transfer 200 ul of culture in a well of a 96-well microplate. | ||
* Measure with or without lid. | * Measure with or without lid. | ||
* Always measure a "blank" that can be subtracted to the other raw measurements (blank for absorbance is sterile media; blank for absorbance is non-fluorescent culture grown to an OD600 comparable to the fluorescent cultures). | * Always measure a "blank" that can be subtracted to the other raw measurements (blank for absorbance is sterile media; blank for absorbance is non-fluorescent culture grown to an OD600 comparable to the fluorescent cultures). | ||
| Line 13: | Line 13: | ||
=== Dynamic readings === | === Dynamic readings === | ||
* Grow bacteria under the desired conditions. | * Grow bacteria under the desired conditions. | ||
* Transfer 200 ul of | * Transfer 200 ul of culture in a well of a 96-well microplate, excluding the 36 wells of the "frame". | ||
* Dispense deionized water in all the unused wells. | * Dispense deionized water in all the unused wells. | ||
* Add inducers to the cultures when required. | * Add inducers to the cultures when required. | ||
* Start a kinetic cycle of 18 hours with the i-control software (Tecan): 37degC during all the experiment, linear shaking (15 s, 3 mm amplitude), 5 s wait time (also 10 s is ok), absorbance reading (default parameters), fluorescence readings (gain=50 for medium and high copy plasmids, gain=80 for low copy plasmids, gain=80 or 100 for single-copy integrants), sampling time=5 min. The specified fluorescence gain factors are only guidelines and can be modified according to promoter/RBS strength, strain and cell density. | * Start a kinetic cycle of 18 hours with the i-control software (Tecan): 37degC during all the experiment, linear shaking (15 s, 3 mm amplitude), 5 s wait time (also 10 s is ok), absorbance reading (default parameters), fluorescence readings (gain=50 for medium and high copy plasmids, gain=50 or 80 for low copy plasmids, gain=80 or 100 for single-copy integrants), sampling time=5 min. The specified fluorescence gain factors are only guidelines and can be modified according to promoter/RBS strength, strain, growth medium and cell density. RFP filters are 535/620nm, GFP filters are 485/540nm. | ||
* Always include a "blank" for absorbance and fluorescence, as described before. | * Always include a "blank" for absorbance and fluorescence, as described before. | ||
* The microplate should be covered with lid for dynamic readings >5-6 hours. | |||
=== List of filters === | |||
* for fluorescence (nm/bandwidth): | |||
**620/20 | |||
**560/20 | |||
**540/25 | |||
**535/25 | |||
**485/20 - we have three of this | |||
**430/35 | |||
* for absorbance (nm): | |||
**600 | |||
**340 | |||
*Commonly used filter pairs for fluorescent proteins (EX=excitation filter, EM=emission filter): | |||
**CFP EX:430 EM:485 | |||
**GFP EX:485 EM:540 | |||
**YFP EX:485 EM:560 | |||
**RFP EX:535 EM:620 | |||
Revision as of 11:00, 28 March 2013
Infinite F200 readings
The microplate reader can be used for static or dynamic readings of bacteria cultures growth and fluorescent proteins.
Static readings
- Grow bacteria under the desired conditions.
- Transfer 200 ul of culture in a well of a 96-well microplate.
- Measure with or without lid.
- Always measure a "blank" that can be subtracted to the other raw measurements (blank for absorbance is sterile media; blank for absorbance is non-fluorescent culture grown to an OD600 comparable to the fluorescent cultures).
Dynamic readings
- Grow bacteria under the desired conditions.
- Transfer 200 ul of culture in a well of a 96-well microplate, excluding the 36 wells of the "frame".
- Dispense deionized water in all the unused wells.
- Add inducers to the cultures when required.
- Start a kinetic cycle of 18 hours with the i-control software (Tecan): 37degC during all the experiment, linear shaking (15 s, 3 mm amplitude), 5 s wait time (also 10 s is ok), absorbance reading (default parameters), fluorescence readings (gain=50 for medium and high copy plasmids, gain=50 or 80 for low copy plasmids, gain=80 or 100 for single-copy integrants), sampling time=5 min. The specified fluorescence gain factors are only guidelines and can be modified according to promoter/RBS strength, strain, growth medium and cell density. RFP filters are 535/620nm, GFP filters are 485/540nm.
- Always include a "blank" for absorbance and fluorescence, as described before.
- The microplate should be covered with lid for dynamic readings >5-6 hours.
List of filters
- for fluorescence (nm/bandwidth):
- 620/20
- 560/20
- 540/25
- 535/25
- 485/20 - we have three of this
- 430/35
- for absorbance (nm):
- 600
- 340
- Commonly used filter pairs for fluorescent proteins (EX=excitation filter, EM=emission filter):
- CFP EX:430 EM:485
- GFP EX:485 EM:540
- YFP EX:485 EM:560
- RFP EX:535 EM:620
