Team:HokkaidoU Japan/Notebook/plastic protocols

From 2012.igem.org

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==PHB Protocols==
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==Polymer producing media==
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===Polymer producing media===
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polymer producing media (LB:2%Glc+ 10mM pantothenic acid Ca + Amp100mg/l) 20ml
polymer producing media (LB:2%Glc+ 10mM pantothenic acid Ca + Amp100mg/l) 20ml
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{|class="hokkaidou-table-digestion"
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|2×LB
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|2ÃLB
|10 ml
|10 ml
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|●50% Glucose
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|â50% Glucose
|800 ul
|800 ul
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|●1M pantothenic acid Ca
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|â1M pantothenic acid Ca
|200 ul
|200 ul
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●50%gulcose
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â50%gulcose
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|Glucose
|Glucose
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|10 g....Heat　and stir until it melts.
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|10 g....Heatãand stir until it melts.
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|ROwater
|ROwater
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Filter sterilize.
Filter sterilize.
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●1M pantothenic acid Ca
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â1M pantothenic acid Ca
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|pantothenic acid Ca
|pantothenic acid Ca
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|4.77 g....Heat　and stir until it melts.
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|4.77 g....Heatãand stir until it melts.
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|ROwater
|ROwater
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Filter sterilize.
Filter sterilize.
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==Culture and harvest==
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===Culture and harvest===
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# Preculture transformed media 1.5ml for 10ï½14 hours, 180rpm/30â.
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# Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30â.
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# Preculture transformed media 1.5ml for 10～14 hours, 180rpm/30℃.
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# Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30℃.
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#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, 5,000rpm and remove its supernatant.
#Centrifuge again for 10min, 5,000rpm and remove its supernatant.
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#Freeze in -80℃ for more than 3hours.
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#Freeze in -80â for more than 3hours.
#Freeze-dry for more than 48hours.
#Freeze-dry for more than 48hours.
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==Polymer extraction and purification==
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===Polymer extraction and purification===
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#Move dried up bacteria into test tube.
#Move dried up bacteria into test tube.
#Break up them to separate and add 10ml chloroform.
#Break up them to separate and add 10ml chloroform.
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#Volatilize chloroform by exposing air again.
#Volatilize chloroform by exposing air again.
#Add 5ml MtOH, voltex for a minute, centrifuge (1,500rpm, 10min), and remove the clear layer.
#Add 5ml MtOH, voltex for a minute, centrifuge (1,500rpm, 10min), and remove the clear layer.
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===Preparation for GC/MS===
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==Preparation for GC/MS==
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Mixture for hydrolysis<br>
Mixture for hydrolysis<br>
All operation must be done with bare hand, so put gloves on.<br>
All operation must be done with bare hand, so put gloves on.<br>
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10. Remove 100 ul solution by pipetman.<br>
10. Remove 100 ul solution by pipetman.<br>
11. Supply to GC/MS.<br>
11. Supply to GC/MS.<br>
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Revision as of 18:49, 26 September 2012

Bold text

Contents

PHB Protocols

Polymer producing media

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

2ÃLB 10 ml
â50% Glucose 800 ul
â1M pantothenic acid Ca 200 ul
Amp(100mg/ml) 20 ul
ROwater 8.98 ml

Put 1.5 ml each into the test tube.


â50%gulcose

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

Filter sterilize.


â1M pantothenic acid Ca

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

Filter sterilize.

Culture and harvest

  1. Preculture transformed media 1.5ml for 10ï½14 hours, 180rpm/30â.
  2. Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30â.
  3. Centrifuge for 10min, 5000rpm.
  4. Remove supernatant and add 500ml ROwater and suspend it.
  5. Centrifuge again for 10min, 5,000rpm and remove its supernatant.
  6. Freeze in -80â 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.

Preparation for GC/MS

Mixture for hydrolysis
All operation must be done with bare hand, so put gloves on.
1. Mix each solution in centrifugation tube (10ml).

Sample 250 ul
HCl 100 ul
Ethanol 850 ul

2. Voltex.
3. Heat at 100C for 4 hours (each 30 min voltex).
4. Cool down centrifugation tube in ice.
5. Add solutions as follow.

(1)
0.65M NaOH
0.9M NaCl
1 ml
(2)
250mM Na2HPO4
(store at 4C) 500 ul

6. Voltex for 1 min.
7. Test by pH test paper (about pH 7.0).
8. Centrifugation for 5 min at 1,500rpm.
9. Pipette chloroform solution by Pasteur pipette which stuffs glass wool and is added about 5 teaspoons of Na2SO4. It means the solution is passed on simple column (Dehydration). Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).
10. Remove 100 ul solution by pipetman.
11. Supply to GC/MS.


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