Protocols/Pouring plates: Difference between revisions
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==Procedure== | ==Procedure== | ||
===Mixing and Sterilizing Media=== | |||
#Add dry materials and H2O to a flask sufficiently large to minimize boil-over. (ex.2 L flask for 1 L of media.) | |||
#(optional, really unnecessary) add a magnetic stir bar and stir the media. | |||
#Cover the top of the flask LOOSELY with aluminum foil. | |||
#Autoclave the media on the Fluid Cycle, 30 min, 255 deg C. (Keep in mind autoclave reduces pressure slowly. It will take more time than 30 min to execute the cycle.) | |||
#Remove media to 55 deg. H2O bath or room temp to cool. | |||
#Once media has cooled to ~55 deg. C, add antibiotics, if required. | |||
===Pouring Plates=== | |||
#Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates. | |||
#Using sterile technique, pour the media into the plates. | |||
#*Cover the base of the plate, and then just a bit more after that. | |||
#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle. | |||
#Leave plates to dry and cool for a while (overnight even). | |||
#*It is a good idea to label the stack of plates to indicate antibiotic. | |||
#Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel). | |||
#Label the bags following taping rules: | |||
#*Red tape in back (indicates LB) | |||
#*Tape in front indicates antibiotic (green=Kan, yellow=Amp, more taping/color rules are on the refrigerator in 68-564) | |||
#*Also write name of antibiotic, and concentration, on the front piece of tape. | |||
#Store the labeled bags of plates in the cold room. | |||
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Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol. | Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol. | ||
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==Notes== | ==Notes== | ||
<!-- Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding <nowiki>*'''~~~~''':</nowiki> in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.<br> | <!-- Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding <nowiki>*'''~~~~''':</nowiki> in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.<br> | ||
Revision as of 14:49, 23 December 2011
Abstract
You need plates. Time to pour some.
Materials
For 1 L of Media:
- 10 g of Agar (for 1% Agar plates (w/v))
- media components specific to media of interest
- 1 L ddH2O
- Sterile antibiotic stock solution (if required)
- Sterile Plates
Equipment
- Autoclave
Procedure
Mixing and Sterilizing Media
- Add dry materials and H2O to a flask sufficiently large to minimize boil-over. (ex.2 L flask for 1 L of media.)
- (optional, really unnecessary) add a magnetic stir bar and stir the media.
- Cover the top of the flask LOOSELY with aluminum foil.
- Autoclave the media on the Fluid Cycle, 30 min, 255 deg C. (Keep in mind autoclave reduces pressure slowly. It will take more time than 30 min to execute the cycle.)
- Remove media to 55 deg. H2O bath or room temp to cool.
- Once media has cooled to ~55 deg. C, add antibiotics, if required.
Pouring Plates
- Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates.
- Using sterile technique, pour the media into the plates.
- Cover the base of the plate, and then just a bit more after that.
- Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.
- Leave plates to dry and cool for a while (overnight even).
- It is a good idea to label the stack of plates to indicate antibiotic.
- Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
- Label the bags following taping rules:
- Red tape in back (indicates LB)
- Tape in front indicates antibiotic (green=Kan, yellow=Amp, more taping/color rules are on the refrigerator in 68-564)
- Also write name of antibiotic, and concentration, on the front piece of tape.
- Store the labeled bags of plates in the cold room.
Notes
It might also be good to add an image to show the workflow and timescales for experiment planning.
Specific Protocols
- Endy:Pouring_plates
- IGEM:Brown/2007/Lab_Protocols/Pouring_Plates_2
- Lissa1:Pouring_Plates
- Endy:Pouring_LB_plates_from_prepped_media
- NanoBio:_LB_plates
Discussion
You can discuss this protocol.
