Team:HokkaidoU Japan/Notebook/plastic protocols

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

50% gulcose (Filter sterilized, Heat and stir)

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

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.

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.

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

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.

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.