Team:Washington/Protocols/Optogenetics/LightAppExperiments

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=General Protocol for Light App Experiments=
 
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==Overnight cultures==
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='''General Protocols for Light App Experiments'''=
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__NOTOC__
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#Cast a gel
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== Overnight Cultures ==
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#Place it in gel box in running buffer
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#Inoculate 3 mL culture tube of LB + 0.1M HEPES buffer pH = 8.0 + 6µL spectinomycin, 3µL kanamycin, and 3µL chloramphenicol with glycerol stock or a colony from a plate.
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#Load samples
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#Cover it with aluminium foil and place it in 37°C shaker overnight.
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#Run the gel
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#Image the gel
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== Casting Gels ==
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== Culture Dilutions ==
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#Get overnight culture from shaker and transfer 500 to 1000 µL of culture to a sterile 1.5mL tube.
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#Centrifuge at 13,000 rpm for 1 min.
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#Discard the supernatant in a waste bottle.
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#Resuspend in unbuffered LB - typically 500-1000µL.
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#Repeat the three steps above two more times (three washes, in total).
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#Dilute resuspended cells into unbuffered LB - 1:125 for soft agar. A dilution of 1:625 is sometimes better for other types of agar.
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'''The amount of agarose to use in your gel depends on the DNA in question.  Use the following table as a rough guide:'''
 
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{| border="1" cellpadding="5" cellspacing="0" align="center"
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== Preparing a 96-Well Plate ==
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|+
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#Melt agar in microwave and keep it in 37°C incubator for 20 minutes.
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! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)  
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#Calculate the amount of agar (see Making Agar section below) and add to sterile tube(s).
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|-=
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#Add corresponding amount of antibiotics (1µL/mL chloramphenicol, 2µL/mL spectinomycin, 1µL/mL kanamycin).  
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| align="center"|0.5 || align="center"|1 - 30
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#Mix them without making bubbles.
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|-
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#Using a pipette, add the agar to each well (200µL/well). It takes ~20 minutes to cool down and solidify.
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| align="center"|0.7 || align="center"|0.8 - 12
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#The plate can be saved by later by covering with film or foil and refrigerating at 4°C.
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|-
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| align="center"|1.0 || align="center"|0.5 - 10
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|-
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| align="center"|1.2 || align="center"|0.4 - 7
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|-
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| align="center"|1.5 || align="center"|0.2 - 3
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|}
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# Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox). 
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== 96-Well Plate Culture ==
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# Microwave until the agarose is fully melted.  This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results.  As long as you do not burn the agarose and nothing bubbles over, this step is robust.
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#Do the following steps in low light.
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# '''From here on, a heat protective glove should be used any time the heated flask must be touched!'''
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#Pipet ~10uL diluted culture into wells in a 96-well plate.
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# Let the agarose cool on the bench for ~5 minutes.
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#Cover the plate with aluminium foil.
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# At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose.  This amount will depend on the concentration of the stock solution of the stain. 
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#Wait for the culture to soak in. It takes about 10 minutes.
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# The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling.
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#Program the Android light app and position the plate on tablet.
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# Pour the agarose solution into the gel casting apparatus.  A pipette tip should be used to pop or shove to the side any bubbles.
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#Place them in incubator for 15 hours.
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# After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
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# Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples.
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Adapted thanks to [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].
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== Using small (~2 in.) plates ==
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#Melt agar in microwave and store it in 37°C incubator for ~20 minutes.
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#Pour necessary amount into sterile tube and add corresponding antibiotics. Each dish needs ~7mL agar.
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#Mix diluted bacteria at a ratio of 15:1000 into the agar. (the total dilution is therefore in the range of 1:40,000)
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#Mix without making bubbles.
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#Gently pour the bacteria+agar mixture into petri dishes, preferably in a darker place.
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#Cover plates with aluminium foil and wait for solidification (~25 minutes).
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#Program the Android light app and put the dishes, without lids, upside down on tablet.
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#Place them into 37°C incubator for 15 hours.
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== Making Agar ==
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Example is for 100mL:
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#100mL LB (pH=6.6, 0.1M HEPES), 50mg Ferric Amonium Citrate, 30mg S-gal, 1g agarose or low-melt agarose.
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#Mix and autoclave. Place in 4°C fridge.
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== Making buffered LB (pH = 6.6 or 8.0) ==
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Example is for 100mL:
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#100mL unbufferd LB, 2.383g (0.01mol) HEPES.
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#Adjust pH by using strong base and pH meter. (approximate amount is around 2-3 mL)
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#Mix and autoclave. Place in 4°C fridge.
'''← [[Team:Washington/Protocols|Back to Protocols]]'''
'''← [[Team:Washington/Protocols|Back to Protocols]]'''

Latest revision as of 02:50, 2 October 2012


General Protocols for Light App Experiments

Overnight Cultures

  1. Inoculate 3 mL culture tube of LB + 0.1M HEPES buffer pH = 8.0 + 6µL spectinomycin, 3µL kanamycin, and 3µL chloramphenicol with glycerol stock or a colony from a plate.
  2. Cover it with aluminium foil and place it in 37°C shaker overnight.


Culture Dilutions

  1. Get overnight culture from shaker and transfer 500 to 1000 µL of culture to a sterile 1.5mL tube.
  2. Centrifuge at 13,000 rpm for 1 min.
  3. Discard the supernatant in a waste bottle.
  4. Resuspend in unbuffered LB - typically 500-1000µL.
  5. Repeat the three steps above two more times (three washes, in total).
  6. Dilute resuspended cells into unbuffered LB - 1:125 for soft agar. A dilution of 1:625 is sometimes better for other types of agar.


Preparing a 96-Well Plate

  1. Melt agar in microwave and keep it in 37°C incubator for 20 minutes.
  2. Calculate the amount of agar (see Making Agar section below) and add to sterile tube(s).
  3. Add corresponding amount of antibiotics (1µL/mL chloramphenicol, 2µL/mL spectinomycin, 1µL/mL kanamycin).
  4. Mix them without making bubbles.
  5. Using a pipette, add the agar to each well (200µL/well). It takes ~20 minutes to cool down and solidify.
  6. The plate can be saved by later by covering with film or foil and refrigerating at 4°C.


96-Well Plate Culture

  1. Do the following steps in low light.
  2. Pipet ~10uL diluted culture into wells in a 96-well plate.
  3. Cover the plate with aluminium foil.
  4. Wait for the culture to soak in. It takes about 10 minutes.
  5. Program the Android light app and position the plate on tablet.
  6. Place them in incubator for 15 hours.


Using small (~2 in.) plates

  1. Melt agar in microwave and store it in 37°C incubator for ~20 minutes.
  2. Pour necessary amount into sterile tube and add corresponding antibiotics. Each dish needs ~7mL agar.
  3. Mix diluted bacteria at a ratio of 15:1000 into the agar. (the total dilution is therefore in the range of 1:40,000)
  4. Mix without making bubbles.
  5. Gently pour the bacteria+agar mixture into petri dishes, preferably in a darker place.
  6. Cover plates with aluminium foil and wait for solidification (~25 minutes).
  7. Program the Android light app and put the dishes, without lids, upside down on tablet.
  8. Place them into 37°C incubator for 15 hours.


Making Agar

Example is for 100mL:

  1. 100mL LB (pH=6.6, 0.1M HEPES), 50mg Ferric Amonium Citrate, 30mg S-gal, 1g agarose or low-melt agarose.
  2. Mix and autoclave. Place in 4°C fridge.


Making buffered LB (pH = 6.6 or 8.0)

Example is for 100mL:

  1. 100mL unbufferd LB, 2.383g (0.01mol) HEPES.
  2. Adjust pH by using strong base and pH meter. (approximate amount is around 2-3 mL)
  3. Mix and autoclave. Place in 4°C fridge.

← Back to Protocols

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