"
Team:HokkaidoU Japan/Notebook/plastic protocols
From 2012.igem.org
(→Polymer extraction and purification) |
|||
| Line 58: | Line 58: | ||
===Culture and harvest=== | ===Culture and harvest=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| - | # Preculture transformed media 1.5 ml for 10~14 | + | # Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C. |
| - | # Culture 15 ul preculture media in polymer producing media for 48 | + | # Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C. |
# Centrifuge for 10 min, 5,000 rpm. | # Centrifuge for 10 min, 5,000 rpm. | ||
# Remove supernatant and add 500 ml RO water and suspend it. | # Remove supernatant and add 500 ml RO water and suspend it. | ||
# Centrifuge again for 10 min, 5,000 rpm and remove its supernatant. | # Centrifuge again for 10 min, 5,000 rpm and remove its supernatant. | ||
| - | # Freeze in -80C for more than 3 | + | # Freeze in -80C for more than 3 hrs. |
| - | # Freeze-dry for more than 48 | + | # Freeze-dry for more than 48 hrs. |
</div> | </div> | ||
| Line 98: | Line 98: | ||
2. Voltex.<br> | 2. Voltex.<br> | ||
| - | 3. Heat at 100C for 4 | + | 3. Heat at 100C for 4 hrs (each 30 min voltex).<br> |
4. Cool down centrifugation tube in ice.<br> | 4. Cool down centrifugation tube in ice.<br> | ||
5. Add solutions as follow.<br> | 5. Add solutions as follow.<br> | ||
Revision as of 03:54, 27 September 2012
Bold text
Contents |
PHB Protocols
Polymer producing media
polymer producing media (LB: 2% Glc, 10 mM pantothenic acid Ca, 100 ug/ml Ampicillin) 20 ml
| 2x LB | 10 ml |
| 50% Glucose | 800 ul |
| 1 M pantothenic acid Ca | 200 ul |
| Amp(100 mg/ml) | 20 ul |
| RO water (autoclaved) | 8.98 ml |
50% gulcose (Filter sterilized, Heat and stir)
| RO water | 7 ml |
| Glucose | 10 g |
| RO water | up to 20 ml |
1 M pantothenic acid Ca (Filter sterilized, Heat and stir)
| RO water | 7 ml |
| pantothenic acid Ca | 4.77 g |
| RO water | up to 10 ml |
Culture and harvest
- Preculture transformed media 1.5 ml for 10~14 hrs, 180 rpm/ 30C.
- Culture 15 ul preculture media in polymer producing media for 48 hrs, 180 rpm/ 30C.
- Centrifuge for 10 min, 5,000 rpm.
- Remove supernatant and add 500 ml RO water and suspend it.
- Centrifuge again for 10 min, 5,000 rpm and remove its supernatant.
- Freeze in -80C for more than 3 hrs.
- Freeze-dry for more than 48 hrs.
Polymer extraction and purification
- Move dried up bacteria into test tube.
- Break up them to separate and add 10 ml chloroform.
- Incubate for 48 hrs at 60C.
- Make it filtered through PTFE and move it into another test tube.
- Volatilize organic solvent by exposing air and separate polymer.
- Add 5 ml hexane and voltex for a minute. After centrifuging (1,500 rpm, 10 min), remove the clear layer.
- Volatilize chloroform by exposing air again.
- Add 5 ml MtOH, voltex for a minute, centrifuge (1,500 rpm, 10 min), 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 hrs (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.
