From 2013.igem.org
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Revision as of 14:06, 24 September 2013
Sep 11
PCR
Tatsui
| pKaiBC(200p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
| pKaiBC(400p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 1 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
| pKaiBC(800p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 2 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
| pKaiBC(1ng) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total
|
| 2.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 98°C | 60°C | 68°C | 30
|
| 2min | 10sec | 30sec | 30sec |
|
colony PCR
Tatsui
| Sample | base pair
|
| 9/9Pcon-lacZα(p3B4k5) | 712bp
|
| 9/9Pcon-spinach-DT(p3B4k5) | 605bp
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
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| 5min | 30s | 30s | 42s |
|
| Sample | base pair
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| Pbad\araC-RBS-luxI-DT |
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| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 5min | 30s | 30s | 2min |
|
| Sample | base pair
|
| 9/9Pcon+RBS-luxI-DT | 1079bp
|
| 9/10Pcon+sptamer12_1R-DT | 584bp
|
| 9/10DT+Pcon-pT181 attenuater 1 | 781bp
|
| 9/10Fusion1 attenuater(p3B1C3)-1 | 638bp
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 55°C | 68°C | --
|
| 50sec | 30sec | 30sec | 1min |
|
Restriction Enzyme Digestion
Nakamoto
| | DNA | EcoRl | Xbal | Spel | Pstl | buffer | BSA | MilliQ | Total |
|
| pSB4K5(EcoRl+Spel) | 9.4µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12.6µL | 30 µL
|
| pSB4K5(EcoRl+Spel) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL
|
| RBS-lacZα-DT(EcoRl+Spel) | 10µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12µL | 30µL
|
| RBS-lacZα-DT(EcoRl+Spel) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL
|
| Ptet-pT181 antisense(Stel+Pstl) | 10.8µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL
|
| Ptet-pT181 antisense(Stel+Pstl) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL
|
| Pcon-RBS-lacZα-DT(EcoRl+Spel) | 7.1µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 14.9µL | 30µL
|
| Pcon-RBS-lacZα-DT(EcoRl+Spel) negative | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL
|
| RBS-lysis2-DT(Xbal+Pstl) | 22µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 0µL | 30µL
|
| RBS-lysis2-DT(Xbal+Pstl) negative | 1.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.6µL | 10µL
|
Liquid Culture
Nakamoto
| Sample | medium
|
| 8/16 Master4 Plax |
|
| 8/25 Pλ-laxI-2 |
|
PCR
Hirano
| 8/29 Pλ-RBS-luxI-DT(76.5ng/µL) | 10x buffer for KOD plus-ver.2 | KOD-plus | dNTP | MgSO4 | F primer(RBS-luxI-DT-cloning-fwd) | R primer(RBS-luxI-DT-cloning-rev) | MilliQ | total
|
| 0.3 | 2.5 | 0.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.2 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 65°C | 68°C | --
|
| 5min | 30sec | 30sec | 1min | 30
|
| 8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL) | 10x buffer for KOD plus-ver.2 | KOD-plus | dNTP | MgSO4 | F primer(Pbad/araC(pSBlc3)-cloning-fwd) | R primer(Pbad/araC(pSBlc3)-cloning-rev) | MilliQ | total
|
| 0.3 | 2.5 | 0.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.2 | 25
|
| PreDenature | Denature | Annealing | Extension | cycle
|
| 94°C | 94°C | 54°C | 68°C | --
|
| 5min | 30sec | 30sec | 3min 20sec | 30
|
Liquid Culture
Hirano
| Sample | medium
|
| 9/4 RBS-lysis1+DT 3 | plusgrow(+CP)
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| 9/4 RBS-lysis2+DT 1 | plusgrow(+CP)
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| 9/4 RBS-lysis3+DT 3 | plusgrow(+CP)
|
===Miniprep===
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