How To Make A MEGA-plate: Difference between revisions
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This page provides step-by-step instructions for the MEGA-Plate experiment for evolving and tracking microbial evolution on antibiotic gradients (Baym, et al., Science, 2016b) | |||
This guide is divided into sections: | |||
==Design of a typical MEGA-plate experiment== | |||
* Example design of regions on the plate (Here for example: 6-step Chloramphenicol gradient experiment). | |||
[[Image:plate_plan1_1.jpg]] | |||
==Assembly of a MEGA-plate== | |||
* Polycarbonate sheet | ===Materials (for a 62x82cm plate)=== | ||
* | * Polycarbonate for the base of the plate: | ||
* | ** Plexiglas sheet 30cm X 60cm X 0.3cm | ||
* Polycarbonate for the walls of the plate | |||
* | ** Polycarbonate sheet 62cm X 82cm X 0.6cm | ||
* Needle | * Heated glass for the lid | ||
* 9 | ** Alternatively acrylic sheet for the lid (superior optical clarity to polycarbonate) | ||
* Sci-Grip adhesive (if not available, Dichloromethane works -- you just need an appropriate solvent for welding acrylics) | |||
* Syringe and Needle- For spreading the glue directly on the right area. | |||
===Procedure=== | |||
==Preparing an appropriate space for imaging== | |||
==Setting up a MEGA-plate experiment== | |||
===Materials=== | |||
* 9 two-liter jars or Erlenmeyer flasks (appropriate for autoclaving) | |||
* Agar (http://www.bd.com/ds/productCenter/214010.asp#) | * Agar (http://www.bd.com/ds/productCenter/214010.asp#) | ||
* LB Broth (http://www.bd.com/ds/productCenter/240220.asp) | * LB Broth (http://www.bd.com/ds/productCenter/240220.asp) | ||
* Appropriate pipettes for adding antibiotics to media | |||
* Blowtorch (e.g. a creme brûlée torch) | |||
* Useful, but not strictly necessary | |||
** IR touchless thermometer | |||
===Procedure=== | |||
* Sanitize the plate by filling it with a dilute bleach solution (~5-10%) overnight | |||
* Dump out bleach solution, do not wipe out residual traces. | |||
** Optional: further sanitize with UV light for around 1 hour. (This is not clearly necessary, and some plastics can leach out reactive compounds when exposed to UV.) | |||
* Prepare 9 liters of agar, either in 2L jars or Erlenmeyer flasks: | |||
** 5 of them with 2L with 2% LB+Agar. | |||
** 2 of them with 2L with 2% Agar. | |||
** 2 of them with 2L with 0.27% Agar. | |||
* Do autoclave 40 min in 121c twice. | |||
* When the bottles are cooling put the plate in the designate place. | |||
* After the liquids are cold enough add the antibiotics. | |||
* Pour the first layer to the right well and let it solidify. | |||
* Pour the second layer all over the plate and let it solidify. | |||
* Pour the third layer all over the plate and let it solidify. | |||
* Inoculate the bacteria. | |||
==Sampling from and disposing of a MEGA-plate experiment== | |||
==Potential failure modes and troubleshooting== | |||
==Procedure for building the plate== | |||
* Use a table saw to cut the frame and the bottom of the plate from the XXX. | |||
[[Image:cut_part1_1.jpg]] [[Image:cut_part1_2.jpg]] | |||
* Use laser cutter to cut the gradient barriers for your design: | |||
XXX | |||
** 8 pieces of 40cm X 1.27cm | |||
** 4 pieces of 40cm X 2cm | |||
** 2 pieces of 60cm X 2cm | |||
[[Image:lazer_cuter.jpg]] [[Image:cut_part2.jpg]] | |||
* Glue the By using syringe and needle glue the pieces like the plan design: | |||
Draw the design of the plate on it. | |||
[[Image:glued_plate2.jpg]] [[Image:glue_plate_1.jpg]] | |||
Put the cut parts in the right place. | |||
[[Image:glued_plate3.jpg]] | |||
Glue them by syringe and needle containing dichloromethane. | |||
[[Image:glued_plate4_1.jpg]] [[Image:glued_plate4_2.jpg]] | |||
* Check for leaking and fix if needed by re-gluing. | |||
==Procedure for imaging the plate== | |||
* Should you wish to take time-lapse movies, we have had success imaging at regular 10-15 minute intervals for the duration of the experiment | |||
==Notes== | ==Notes== | ||
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | ||
#List troubleshooting tips here. | #List troubleshooting tips here. | ||
==References== | ==References== | ||
'''Relevant papers and books''' | '''Relevant papers and books''' | ||
# Baym, M., Lieberman, T. D., Kelsic, E. D., Chait, R., Gross, R., Yelin, I., & Kishony, R. (2016). Spatiotemporal microbial evolution on antibiotic landscapes. Science, 353(6304), 1147-1151. doi:10.1126/science.aag0822[http://science.sciencemag.org/content/353/6304/1147] | |||
==Contact== | ==Contact== | ||
This page was developed by Michael Baym and Rotem Gross from the Kishony Lab. | |||
For questions, please contact: | |||
# Michael Baym (mhbaym@gmail.com) | |||
# Rotem Gross (rotemgross1@gmail.com) | |||
# Roy Kishony (rkishony@technion.ac.il) | |||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> |
Latest revision as of 00:54, 8 June 2017
This page provides step-by-step instructions for the MEGA-Plate experiment for evolving and tracking microbial evolution on antibiotic gradients (Baym, et al., Science, 2016b)
This guide is divided into sections:
Design of a typical MEGA-plate experiment
- Example design of regions on the plate (Here for example: 6-step Chloramphenicol gradient experiment).
Assembly of a MEGA-plate
Materials (for a 62x82cm plate)
- Polycarbonate for the base of the plate:
- Plexiglas sheet 30cm X 60cm X 0.3cm
- Polycarbonate for the walls of the plate
- Polycarbonate sheet 62cm X 82cm X 0.6cm
- Heated glass for the lid
- Alternatively acrylic sheet for the lid (superior optical clarity to polycarbonate)
- Sci-Grip adhesive (if not available, Dichloromethane works -- you just need an appropriate solvent for welding acrylics)
- Syringe and Needle- For spreading the glue directly on the right area.
Procedure
Preparing an appropriate space for imaging
Setting up a MEGA-plate experiment
Materials
- 9 two-liter jars or Erlenmeyer flasks (appropriate for autoclaving)
- Agar (http://www.bd.com/ds/productCenter/214010.asp#)
- LB Broth (http://www.bd.com/ds/productCenter/240220.asp)
- Appropriate pipettes for adding antibiotics to media
- Blowtorch (e.g. a creme brûlée torch)
- Useful, but not strictly necessary
- IR touchless thermometer
Procedure
- Sanitize the plate by filling it with a dilute bleach solution (~5-10%) overnight
- Dump out bleach solution, do not wipe out residual traces.
- Optional: further sanitize with UV light for around 1 hour. (This is not clearly necessary, and some plastics can leach out reactive compounds when exposed to UV.)
- Prepare 9 liters of agar, either in 2L jars or Erlenmeyer flasks:
- 5 of them with 2L with 2% LB+Agar.
- 2 of them with 2L with 2% Agar.
- 2 of them with 2L with 0.27% Agar.
- Do autoclave 40 min in 121c twice.
- When the bottles are cooling put the plate in the designate place.
- After the liquids are cold enough add the antibiotics.
- Pour the first layer to the right well and let it solidify.
- Pour the second layer all over the plate and let it solidify.
- Pour the third layer all over the plate and let it solidify.
- Inoculate the bacteria.
Sampling from and disposing of a MEGA-plate experiment
Potential failure modes and troubleshooting
Procedure for building the plate
- Use a table saw to cut the frame and the bottom of the plate from the XXX.
- Use laser cutter to cut the gradient barriers for your design:
XXX
- 8 pieces of 40cm X 1.27cm
- 4 pieces of 40cm X 2cm
- 2 pieces of 60cm X 2cm
- Glue the By using syringe and needle glue the pieces like the plan design:
Draw the design of the plate on it.
Put the cut parts in the right place.
Glue them by syringe and needle containing dichloromethane.
- Check for leaking and fix if needed by re-gluing.
Procedure for imaging the plate
- Should you wish to take time-lapse movies, we have had success imaging at regular 10-15 minute intervals for the duration of the experiment
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
References
Relevant papers and books
- Baym, M., Lieberman, T. D., Kelsic, E. D., Chait, R., Gross, R., Yelin, I., & Kishony, R. (2016). Spatiotemporal microbial evolution on antibiotic landscapes. Science, 353(6304), 1147-1151. doi:10.1126/science.aag0822[1]
Contact
This page was developed by Michael Baym and Rotem Gross from the Kishony Lab.
For questions, please contact:
- Michael Baym (mhbaym@gmail.com)
- Rotem Gross (rotemgross1@gmail.com)
- Roy Kishony (rkishony@technion.ac.il)