Endy:Pouring plates: Difference between revisions

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This is a general protocol is for the standard preparation of plates.
Also see the [[Endy:Pouring LB plates from prepped media|shortcut method]] for using pre-made 1.2% Agar in LB from the Building 68 media prep room.
==Notes==
==Notes==


*Melt LB ahead of time because it cools to 55 degrees slowly. A good way to do this is melt it in the morning then leave it in the 60 degree incubator or a 60 degree bath. If in a rush, do not attempt to cool the media rapidly by running cold water over the container as this can cause the glass to shatter.  Instead, media can be left at room temperature to cool more rapidly.
*(Optional) pre-heat a H2O bath to 55 deg C.  
*If you don't know how to use the autoclave, ask for a demo.   


==Materials==
==Materials==
 
For 1 L of Media:
*LB Agar 1.2% (from the media room)
*10 g of Agar (for 1% Agar plates (w/v))
*Empty plates (above any of the -20 freezers)
*media components specific to media of interest
*antibiotics (liquid stocks from our -20 freezer)
*1 L ddH2O
*Sterile antibiotic stock solution (if required)


==Method==
==Method==
===Preparation of LB Agar===
===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.)
#Obtain 1.2% LB agar from the media room (500 ml per bottle).
#(optional, really unnecessary) add a magnetic stir bar and stir the media.
#*Be sure to sign it out
#Cover the top of the flask LOOSELY with aluminum foil.
#Melt in microwave:
#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.)
#*loosen the cap
#Remove media to 55 deg. H2O bath or room temp to cool.
#*use 50% power (enter time, press Power, 5, Start)
#Once media has cooled to ~55 deg. C, add antibiotics, if required.
#*monitor as you melt
#*takes approx. 10 minutes per bottle.
#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.
#Once it's cool enough (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar use:
#*500μL Amp stock
#*1mL Kan stock
#Swirl to mix; try not to make many bubbles.


===Actual Pouring===
===Pouring Plates===


#Obtain a container of empty plates. One bottle (500ml) of LB Agar will make about one container of plates (20 plates).
#Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates.
#Using sterile technique (flame the top of the bottle)(why? what are you killing here? the plastic sealing ring?), pour the LB Agar into the plates.  
#Using sterile technique, pour the media into the plates.  
#*Cover the base of the plate, and then just a bit more after that.
#*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.
#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.

Revision as of 12:37, 19 June 2006

This is a general protocol is for the standard preparation of plates.

Also see the shortcut method for using pre-made 1.2% Agar in LB from the Building 68 media prep room.

Notes

  • (Optional) pre-heat a H2O bath to 55 deg C.
  • If you don't know how to use the autoclave, ask for a demo.

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)

Method

Mixing and Sterilizing Media

  1. Add dry materials and H2O to a flask sufficiently large to minimize boil-over. (ex.2 L flask for 1 L of media.)
  2. (optional, really unnecessary) add a magnetic stir bar and stir the media.
  3. Cover the top of the flask LOOSELY with aluminum foil.
  4. 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.)
  5. Remove media to 55 deg. H2O bath or room temp to cool.
  6. Once media has cooled to ~55 deg. C, add antibiotics, if required.

Pouring Plates

  1. Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates.
  2. Using sterile technique, pour the media into the plates.
    • Cover the base of the plate, and then just a bit more after that.
  3. 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.
  4. 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.
  5. Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
  6. 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.
  7. Store the labeled bags of plates in the cold room.
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