Team:HokkaidoU Japan/Notebook/plastic protocols

From 2012.igem.org

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==PHB Protocols==
-
==Polymer producing media==
+
===Polymer producing media===
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<div>
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<div class="hokkaidou-section">
-
<p>
+
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml
-
polymer producing media (LB:2%Glc+ 10mM pantothenic acid Ca + Amp100mg/l) 20ml
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{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
|-
|-
-
|2×LB
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|2x LB
-
|10ml
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|10 ml
|-
|-
-
|●50% Glucose
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|50% Glucose
-
|800ul
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|800 ul
|-
|-
-
|●1M pantothenic acid Ca
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|1 M pantothenic acid Ca
-
|200ul
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|200 ul
|-
|-
-
|Amp(100mg/ml)
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|Amp(100 mg/ml)
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|20ul
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|20 ul
|-
|-
-
|ROwater
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|RO water (autoclaved)
-
|8.98ml
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|8.98 ml
|-
|-
|}
|}
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Put 1.5ml each into the test tube.
 
-
 
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50% gulcose (Filter sterilized, Heat and stir)
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●50%gulcose
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{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
|-
|-
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|ROwater
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|RO water
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|7ml
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|7 ml
|-
|-
|Glucose
|Glucose
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|10g....Heat　and stir until it melts.
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|10 g
|-
|-
-
|ROwater
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|RO water
-
|up to 20ml
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|up to 20 ml
|-
|-
|}
|}
-
Filter sterilize.
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1 M pantothenic acid Ca (Filter sterilized, Heat and stir)
-
 
+
-
 
+
-
●1M pantothenic acid Ca
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{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
|-
|-
-
|ROwater
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|RO water
-
|7ml
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|7 ml
|-
|-
|pantothenic acid Ca
|pantothenic acid Ca
-
|4.77g....Heat　and stir until it melts.
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|4.77 g
|-
|-
-
|ROwater
+
|RO water
-
|up to 10ml
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|up to 10 ml
|-
|-
|}
|}
 +
</div>
-
Filter sterilize.
+
===Culture and harvest===
-
 
+
<div class="hokkaidou-section">
-
 
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# Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.
-
 
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# Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.
-
</p>
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# Centrifuge for 10 min, 5,000 rpm.
 +
# Remove supernatant and add 500 ml RO water and suspend it.
 +
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.
 +
# Freeze in -80C for more than 3 hrs.
 +
# Freeze-dry for more than 48 hrs.
</div>
</div>
-
==Culture and harvest==
+
===Polymer extraction and purification===
-
<div>
+
<div class="hokkaidou-section">
-
<p>
+
# Move dried up bacteria into test tube.
-
 
+
# Break up them to separate and add 10 ml chloroform.
-
# Preculture transformed media 1.5ml for 10～14 hours, 180rpm/30℃.
+
# Incubate for 48 hrs at 60C.
-
# Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30℃.
+
# Make it filtered through PTFE and move it into another test tube.
-
#Harvest by centrifuge for 10min, 5000rpm.
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# Volatilize organic solvent by exposing air and separate polymer.
-
#Remove supernatant and add 500ml ROwater and suspend it.
+
# Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.
-
#Centrifuge again for 10min, 5000rpm and remove its supernatant.
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# Volatilize chloroform by exposing air again.
-
#Freeze in -80℃ for more than 3hours.
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# Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.
-
#Freeze-dry for more than 48hours.
+
-
 
+
-
</p>
+
</div>
</div>
-
==Polymer extraction and purification==
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===Preparation for GC/MS===
-
<div>
+
<div class="hokkaidou-section">
-
<p>
+
Mixture for hydrolysis<br />
-
#Move dried up bacteria into test tube.
+
All operation must be done with bare hand, so put gloves on.<br />
-
#Break up them to separate and add 10ml chloroform.
+
1. Mix each solution in centrifugation tube (10 ml).
-
#Incubate for 48hour at 60C.
+
{|class="hokkaidou-table-digestion"
-
#Make it filtered through PTFE and move it into another test tube.
+
|-
-
#Volatilize organic solvent by exposing air and separate polymer.
+
|Sample
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#Add 5ml hexane and voltex for a minute. After centrifuging (1,500rpm, 10min), remove the clear layer.
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|250 ul
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#Volatilize chloroform by exposing air again.
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|-
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#Add 5ml MtOH, voltex for a minute, centrifuge (1,500rpm, 10min), and remove the clear layer.
+
|HCl
 +
|100 ul
 +
|-
 +
|Ethanol
 +
|850 ul
 +
|-
 +
|}
 +
2. Voltex.<br />
 +
3. Heat at 100C for 4 hrs (each 30 min voltex).<br />
 +
4. Cool down centrifugation tube in ice.<br />
 +
5. Add solutions as follow.<br />
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|(1)
 +
|-
 +
|0.65M NaOH
 +
|
 +
|-
 +
|0.9M NaCl
 +
|
 +
|-
 +
|
 +
|1 ml
 +
|-
 +
|(2)
 +
|-
 +
|250mM Na2HPO4
 +
|-
 +
|(store at 4C)
 +
| 500 ul
 +
|-
 +
|}
-
 
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6. Voltex for 1 min.<br />
-
</p>
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7. Test by pH test paper (about pH 7.0).<br />
-
</div></div>
+
8. Centrifugation for 5 min at 1,500 rpm.<br />
 +
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).<br />
 +
10. Remove 100 ul solution by pipetman.<br />
 +
11. Supply to GC/MS.<br />
 +
</div>
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Latest revision as of 03:56, 27 September 2012

Bold text

Contents

PHB Protocols

Polymer producing media

polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml

2x LB 10 ml
50% Glucose 800 ul
1 M pantothenic acid Ca 200 ul
Amp(100 mg/ml) 20 ul
RO water (autoclaved) 8.98 ml

50% gulcose (Filter sterilized, Heat and stir)

RO water 7 ml
Glucose 10 g
RO water up to 20 ml

1 M pantothenic acid Ca (Filter sterilized, Heat and stir)

RO water 7 ml
pantothenic acid Ca 4.77 g
RO water up to 10 ml

Culture and harvest

  1. Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.
  2. Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.
  3. Centrifuge for 10 min, 5,000 rpm.
  4. Remove supernatant and add 500 ml RO water and suspend it.
  5. Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.
  6. Freeze in -80C for more than 3 hrs.
  7. Freeze-dry for more than 48 hrs.

Polymer extraction and purification

  1. Move dried up bacteria into test tube.
  2. Break up them to separate and add 10 ml chloroform.
  3. Incubate for 48 hrs 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 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.
  7. Volatilize chloroform by exposing air again.
  8. Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), 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 (10 ml).

Sample 250 ul
HCl 100 ul
Ethanol 850 ul

2. Voltex.
3. Heat at 100C for 4 hrs (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 1,500 rpm.
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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