Team:HokkaidoU Japan/Notebook/plastic protocols

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

Put 1.5 ml each into the test tube.

â50%gulcose

Filter sterilize.

â1M pantothenic acid Ca

Filter sterilize.

1. Preculture transformed media 1.5ml for 10ï½14 hours, 180rpm/30â.
2. Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30â.
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 -80â for more than 3hours.
7. Freeze-dry for more than 48hours.

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.

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.