Team:HokkaidoU Japan/Notebook/plastic protocols

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

50% gulcose (Filter sterilized)

1 M pantothenic acid Ca (Filter sterilized)

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.

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.

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

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.

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.