Team:HokkaidoU Japan/Notebook/plastic protocols

From 2012.igem.org

(Difference between revisions)
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===Polymer producing media===
===Polymer producing media===
<div class="hokkaidou-section">
<div class="hokkaidou-section">
-
polymer producing media (LB:2%Glc+ 10mM pantothenic acid Ca + Amp100mg/l) 20ml
+
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100ug/ml Ampicillin) 20ml
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
|-
|-
-
|2ÃLB
+
|2x LB
|10 ml
|10 ml
|-
|-
-
|â50% Glucose
+
|50% Glucose
|800 ul
|800 ul
|-
|-
-
|â1M pantothenic acid Ca
+
|1 M pantothenic acid Ca
|200 ul
|200 ul
|-
|-
-
|Amp(100mg/ml)
+
|Amp(100 mg/ml)
|20 ul
|20 ul
|-
|-
-
|ROwater
+
|RO water (autoclaved)
|8.98 ml
|8.98 ml
|-
|-
|}
|}
-
Put 1.5 ml each into the test tube.
 
-
 
+
50% gulcose (Filter sterilized)
-
â50%gulcose
+
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
|-
|-
-
|ROwater
+
|RO water
|7 ml
|7 ml
|-
|-
|Glucose
|Glucose
-
|10 g....Heatãand stir until it melts.
+
|10 g....Heat and stir until it melts.
|-
|-
-
|ROwater
+
|RO water
|up to 20 ml
|up to 20 ml
|-
|-
|}
|}
-
Filter sterilize.
 
-
 
-
â1M pantothenic acid Ca
+
1 M pantothenic acid Ca (Filter sterilized)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
|-
|-
-
|ROwater
+
|RO water
|7 ml
|7 ml
|-
|-
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|4.77 g....Heatãand stir until it melts.
|4.77 g....Heatãand stir until it melts.
|-
|-
-
|ROwater
+
|RO water
|up to 10 ml
|up to 10 ml
|-
|-
|}
|}
-
Filter sterilize.
 
</div>
</div>
===Culture and harvest===
===Culture and harvest===
<div class="hokkaidou-section">
<div class="hokkaidou-section">
-
# Preculture transformed media 1.5ml for 10ï½14 hours, 180rpm/30â.
+
# Preculture transformed media 1.5 ml for 10~14 hours, 180rpm/30C.
-
# Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30â.
+
# Culture 15 ul preculture media in polymer producing media for 48hours, 180rpm/30C.
-
#Centrifuge for 10min, 5000rpm.
+
# Centrifuge for 10 min, 5000 rpm.
-
#Remove supernatant and add 500ml ROwater and suspend it.
+
# Remove supernatant and add 500 ml RO water and suspend it.
-
#Centrifuge again for 10min, 5,000rpm and remove its supernatant.
+
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.
-
#Freeze in -80â for more than 3hours.
+
# Freeze in -80C for more than 3 hours.
-
#Freeze-dry for more than 48hours.
+
# Freeze-dry for more than 48 hours.
</div>
</div>
===Polymer extraction and purification===
===Polymer extraction and purification===
<div class="hokkaidou-section">
<div class="hokkaidou-section">
-
#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 10 ml chloroform.
-
#Incubate for 48hour at 60C.
+
# Incubate for 48 hour at 60C.
-
#Make it filtered through PTFE and move it into another test tube.
+
# Make it filtered through PTFE and move it into another test tube.
-
#Volatilize organic solvent by exposing air and separate polymer.
+
# Volatilize organic solvent by exposing air and separate polymer.
-
#Add 5ml hexane and voltex for a minute. After centrifuging (1,500rpm, 10min), remove the clear layer.
+
# Add 5ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.
-
#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 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), and remove the clear layer.
</div>
</div>

Revision as of 22:59, 26 September 2012

Bold text

Contents

PHB Protocols

Polymer producing media

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

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)

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

1 M pantothenic acid Ca (Filter sterilized)

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

Culture and harvest

  1. Preculture transformed media 1.5 ml for 10~14 hours, 180rpm/30C.
  2. Culture 15 ul preculture media in polymer producing media for 48hours, 180rpm/30C.
  3. Centrifuge for 10 min, 5000 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 hours.
  7. Freeze-dry for more than 48 hours.

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 hour 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 (1,500 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 (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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