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Template:Kyoto/Notebook/Sep 11
From 2013.igem.org
Contents |
Sep 11
PCR
| 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
| 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 | -- |
| 5min | 30s | 30s | 42s |
| Sample | base pair |
|---|---|
| Pbad\araC-RBS-luxI-DT |
| 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 |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | 9/8pSB4K5(EcoRl&Spel)21.8ng/µL | 2.3 µL | 9/8Pcon-Spinach-DT(EcoRl&Spel)10.5ng/µL | 1.7 µL | 4.0 µL |
| NC | 9/8pSB4K5(EcoRl&Spel)21.8ng/µL | 2.3 µL | 9/8RBS-lacZα-DT(EcoRl&Spel)4.4ng/µL | 6.7µL | 9.0 µL |
| NC | 9/8pSB4K5(EcoRl&Spel)21.8ng/µL | 2.3 µL | 9/8Pcon-RBS-lacZα-DT(EcoRl&Spel)5.6ng/µL | 1.9 µL | 4.2 µL |
| NC | 9/6Plux 25ng/µL | 2 µL | 9/8 RBS-lysis1-DT(Xbal&pstl) | 6.6 µL | 8.6 µL |
| NC | 9/6Plux 25ng/µL | 2 µL | 9/8 RBS-lysis3-DT(Xbal&pstl) | 4.7 µL | 6.7 µL |
| NC | 9/10Pcon-pT181 attenuater(Spel&Pstl) | 3.4µL | 9/8RBS-lacZα-DT(EcoRl&Spel) | 4.5 µL | 7.9 µL |
| NC | 9/6Pcon-RBS laxR-DT(EcoRl&Xbal) 26.7ng/µL | 1.9 µL | Plax-RBS-GFP-DT | 5.5 µL | 7.4 µL |
| NC | 9/9 Pcon-pT181 attenuater(SPpel&Pstl) | xx µL | name | xx µL | xx µL |
Restriction Enzyme Digestion
| 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
| Sample | medium |
|---|---|
| 8/16 Master4 Plax | |
| 8/25 Pλ-laxI-2 |
PCR
| 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
| Sample | medium |
|---|---|
| 9/4 RBS-lysis1+DT 3 | plusgrow(+CP) 3ml 37°C |
| 9/4 RBS-lysis2+DT 1 | plusgrow(+CP) 3ml 37°C |
| 9/4 RBS-lysis3+DT 3 | plusgrow(+CP) 3ml 37°C |
