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Team:EPF-Lausanne/Protocols/Agar Plates
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Agar plates are used for cell cultures. Their preparation is relatively simple: first an agar broth is mixed and autoclaved. Then an antibacterial agent such as Ampicillin is mixed in. Finally, the solution is poured into disposable petri plates and let gelify for 20 minutes around a bunzen burner flame, as illustrated below. | Agar plates are used for cell cultures. Their preparation is relatively simple: first an agar broth is mixed and autoclaved. Then an antibacterial agent such as Ampicillin is mixed in. Finally, the solution is poured into disposable petri plates and let gelify for 20 minutes around a bunzen burner flame, as illustrated below. | ||
| - | [[File:EPFL-Growth_plate_gelification.JPG|600px]] | + | [[File:EPFL-Growth_plate_gelification.JPG|600px|center]] |
== Agar broth == | == Agar broth == | ||
| + | |||
| + | The antibiotic agent will depend on what kind of plasmid is used. Listed here is an example for Ampicillin (used for plasmids carrying the Ampicillin resistance gene). | ||
| + | |||
| + | Mix the following in a large bottle: | ||
| + | |||
| + | {| | ||
| + | |+ Ingredients for 1 l of LB Agar | ||
| + | ! Product || Amount | ||
| + | |- | ||
| + | | NaCl || 10 g | ||
| + | |- | ||
| + | | Tryptone || 10 g | ||
| + | |- | ||
| + | | Yeast Extract || 5 g | ||
| + | |- | ||
| + | | Agar || 20 g | ||
| + | |- | ||
| + | | H2O (deionised) || fill to 1 l | ||
| + | |} | ||
| + | |||
| + | * Adjust pH to 7.0 using 5 M NaOH. | ||
| + | * Autoclave. | ||
| + | * Keep in 55° C bath after autoclaving (that's where it will arrive if autoclaved in Bart's lab). | ||
| + | * Add 100 mg filter-sterilised ampicillin. | ||
| + | * Mix on heating mixer, until homogenous (don't close lid completely). | ||
| + | |||
| + | |||
| + | '''Warning''': the broth starts gelification as soon as it cools down. Store it in a warm bath to keep it liquid. If it does gelify, just warm and mix again (with magnetic mixer). | ||
| + | |||
| + | ''' | ||
== Pouring the plates == | == Pouring the plates == | ||
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Arrange empty petri dishes in a crescent around a Bunzen burner. Half-fill them with agar broth. Stack another layer of empty dishes above, and repeat until all the broth has been poured. One liter of broth fills approximately 30 plates. | Arrange empty petri dishes in a crescent around a Bunzen burner. Half-fill them with agar broth. Stack another layer of empty dishes above, and repeat until all the broth has been poured. One liter of broth fills approximately 30 plates. | ||
| - | Leave the plates around the flame for approximately twenty minutes, until the gel is solidified. | + | Leave the plates around the flame for approximately twenty minutes, until the gel is solidified. Then replace lid and store in fridge. |
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Latest revision as of 15:39, 20 July 2011
Agar plate preparation
Back to protocols.Agar plates are used for cell cultures. Their preparation is relatively simple: first an agar broth is mixed and autoclaved. Then an antibacterial agent such as Ampicillin is mixed in. Finally, the solution is poured into disposable petri plates and let gelify for 20 minutes around a bunzen burner flame, as illustrated below.
Agar broth
The antibiotic agent will depend on what kind of plasmid is used. Listed here is an example for Ampicillin (used for plasmids carrying the Ampicillin resistance gene).
Mix the following in a large bottle:
| Product | Amount |
|---|---|
| NaCl | 10 g |
| Tryptone | 10 g |
| Yeast Extract | 5 g |
| Agar | 20 g |
| H2O (deionised) | fill to 1 l |
- Adjust pH to 7.0 using 5 M NaOH.
- Autoclave.
- Keep in 55° C bath after autoclaving (that's where it will arrive if autoclaved in Bart's lab).
- Add 100 mg filter-sterilised ampicillin.
- Mix on heating mixer, until homogenous (don't close lid completely).
Warning: the broth starts gelification as soon as it cools down. Store it in a warm bath to keep it liquid. If it does gelify, just warm and mix again (with magnetic mixer).
Pouring the plates
Arrange empty petri dishes in a crescent around a Bunzen burner. Half-fill them with agar broth. Stack another layer of empty dishes above, and repeat until all the broth has been poured. One liter of broth fills approximately 30 plates.
Leave the plates around the flame for approximately twenty minutes, until the gel is solidified. Then replace lid and store in fridge.
