Team:HokkaidoU Japan/Notebook/plastic protocols

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Contents

Polymer producing media

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

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

Put 1.5 ml each into the test tube.


●50%gulcose

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

Filter sterilize.


●1M pantothenic acid Ca

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

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, 5,000rpm 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.


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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