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Team:HokkaidoU Japan/Notebook/plastic protocols
From 2012.igem.org
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Mixture for hydrolysis<br> | Mixture for hydrolysis<br> | ||
All operation must be done with bare hand, so put gloves on.<br> | All operation must be done with bare hand, so put gloves on.<br> | ||
| - | + | 1. Mix each solution in centrifugation tube (10ml). | |
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| - | + | 2. Voltex.<br> | |
| - | + | 3. Heat at 100C for 4 hours (each 30 min voltex).<br> | |
| - | + | 4. Cool down centrifugation tube in ice.<br> | |
| - | + | 5. Add solutions as follow.<br> | |
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| - | + | 6. Voltex for 1 min.<br> | |
| - | + | 7. Test by pH test paper (about pH 7.0).<br> | |
| - | + | 8. Centrifugation for 5 min at 1,500rpm.<br> | |
| - | + | 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). | + | It means the solution is passed on simple column (Dehydration). |
| - | Passed solution is collected by vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako) | + | Passed solution is collected by a vial whose bottom is covered by Molecular sieves 4A 1/16 (producted by Wako).<br> |
| - | + | 10. Remove 100 ul solution by pipetman.<br> | |
| - | + | 11. Supply to GC/MS.<br> | |
Revision as of 18:28, 25 September 2012
Bold text
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
- Preculture transformed media 1.5ml for 10～14 hours, 180rpm/30℃.
- Culture 15μl preculture media in polymer producing media for 48hours, 180rpm/30℃.
- Centrifuge for 10min, 5000rpm.
- Remove supernatant and add 500ml ROwater and suspend it.
- Centrifuge again for 10min, 5,000rpm and remove its supernatant.
- Freeze in -80℃ for more than 3hours.
- Freeze-dry for more than 48hours.
Polymer extraction and purification
- Move dried up bacteria into test tube.
- Break up them to separate and add 10ml chloroform.
- Incubate for 48hour at 60C.
- Make it filtered through PTFE and move it into another test tube.
- 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.
- Volatilize chloroform by exposing air again.
- 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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