Team:HokkaidoU Japan/Notebook/plastic protocols

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(→Culture and harvest)
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# Preculture transformed media 1.5ml for 10～14 hours, 180rpm/30℃.
# Preculture transformed media 1.5ml for 10～14 hours, 180rpm/30℃.
# Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30℃.
# Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30℃.
-
#Harvest by centrifuge for 10min, 5000rpm.
+
#Centrifuge for 10min, 5000rpm.
#Remove supernatant and add 500ml ROwater and suspend it.
#Remove supernatant and add 500ml ROwater and suspend it.
#Centrifuge again for 10min, 5000rpm and remove its supernatant.
#Centrifuge again for 10min, 5000rpm and remove its supernatant.

Revision as of 12:13, 6 August 2012

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Polymer producing media

polymer producing media (LB:2%Glc+ 10mM pantothenic acid Ca + Amp100mg/l) 20ml

2×LB 10ml
●50% Glucose 800ul
●1M pantothenic acid Ca 200ul
Amp(100mg/ml) 20ul
ROwater 8.98ml

Put 1.5ml each into the test tube.


●50%gulcose

ROwater 7ml
Glucose 10g....Heat　and stir until it melts.
ROwater up to 20ml

Filter sterilize.


●1M pantothenic acid Ca

ROwater 7ml
pantothenic acid Ca 4.77g....Heat　and stir until it melts.
ROwater up to 10ml

Filter sterilize.


Culture and harvest

  1. Preculture transformed media 1.5ml for 10～14 hours, 180rpm/30℃.
  2. Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30℃.
  3. Centrifuge for 10min, 5000rpm.
  4. Remove supernatant and add 500ml ROwater and suspend it.
  5. Centrifuge again for 10min, 5000rpm and remove its supernatant.
  6. Freeze in -80℃ for more than 3hours.
  7. Freeze-dry for more than 48hours.

Polymer extraction and purification

  1. Move dried up bacteria into test tube.
  2. Break up them to separate and add 10ml chloroform.
  3. Incubate for 48hour at 60C.
  4. Make it filtered through PTFE and move it into another test tube.
  5. Volatilize organic solvent by exposing air and separate polymer.
  6. Add 5ml hexane and voltex for a minute. After centrifuging (1,500rpm, 10min), remove the clear layer.
  7. Volatilize chloroform by exposing air again.
  8. Add 5ml MtOH, voltex for a minute, centrifuge (1,500rpm, 10min), and remove the clear layer.


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