Template:Kyoto/Notebook/Sep 5
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Revision as of 19:23, 25 September 2013
Sep 5
Electrophoresis
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 100bp ladder | -- | -- |
2 | J23100-3 | -- | -- |
3 | J23100-4 | -- | -- |
4 | J23100-5 | -- | -- |
5 | 100bp ladder | -- | -- |
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
8/3 J23100-3 | 349.1 | 1.69 | 1.851 |
8/10 Plac-2 | 331.3 | 1.84 | 1.42 |
Ligation
state | Vector | Inserter | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | Pconst | 13.2 | spinach-DT | 2.1 | 3.5 |
experiment | Plac | 15.3 | spinach-DT | 2.1 | 3.5 |
experiment | Pconst | 13.2 | pT181 antisense (XbaI & PstI) | 6.6 | 3.5 |
experiment | Plac | 15.3 | pT181 antisense (XbaI & PstI) | 6.6 | 3.5 |
experiment | Ptet | 30.1 | pT181 antisense (XbaI & PstI) | 6.6 | 3.5 |
experiment | Pconst | 13.2 | pT181 attenuator | 11.7 | 3.5 |
experiment | Plac | 15.3 | pT181 attenuator | 11.7 | 3.5 |
experiment | Ptet | 30.1 | RBS-lacZα-DT | 3.0 | 3.5 |
incubate 16 °C 1 hour
Electrophoresis
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | |
2 | RBS-luxI-DT | |
3 | RBS-luxI-DT | |
4 | ||
5 | Pbad/araC | |
6 | Pbad/araC |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
RBS-luxI-DT | 23.2 | 0.83 | 7.89 |
Pbad/araC | 51.8 | 1.20 | 0.11 |
Colony PCR
Sample | base pair |
---|---|
9/4 aptamer 12_1R(pSB1C3)-1 | 384 |
9/4 aptamer 12_1R(pSB1C3)-2 | 384 |
9/4 pT181 antisense(pSB1C3)-1 | 415 |
9/4 pT181 antisense(pSB1C3)-2 | 415 |
9/4 pT181 antisense(pSB1C3)-3 | 415 |
9/4 pT181 antisense(pSB1C3)-4 | 415 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 30s | 30cycles |
Sample | base pair |
---|---|
9/4 aptamer 12_1R-DT-1 | 521 |
9/4 aptamer 12_1R-DT-2 | 521 |
9/4 aptamer 12_1R-DT-3 | 521 |
9/4 aptamer 12_1R-DT-4 | 521 |
9/4 pT181 attenuator(pSB1C3) | 601 |
9/4 pT181 attenuator(pSB1C3) | 601 |
9/4 RBS-lysis1-DT | 613 |
9/4 RBS-lysis1-DT | 613 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 36s | 30cycles |
Sample | base pair |
---|---|
9/4 RBS-lysis2-DT-1 | 985 |
9/4 RBS-lysis2-DT-2 | 985 |
9/4 RBS-lysis2-DT-3 | 985 |
9/4 RBS-lysis2-DT-4 | 985 |
9/4 RBS-lysis3-DT-1 | 1210 |
9/4 RBS-lysis3-DT-2 | 1210 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 72s | 30cycles |
Liquid Culture
Sample | medium |
---|---|
8/9 J23100-5 | Plusgrow medium(+Amp) |
37°C
Transformation
Name | Sample(µL) | Competent Cells(µL) | Total(µL) | Plate |
---|---|---|---|---|
9/5 Pconst+spinach-DT | 2 | 20 | 22 | -- |
9/5 Plac+spinach-DT | 2 | 20 | 22 | -- |
9/5 Pconst+pT181 antisense | 2 | 20 | 22 | -- |
9/5 Plac+pT181 antisense | 2 | 20 | 22 | -- |
9/5 Ptet+pT181 antisense | 2 | 20 | 22 | -- |
9/5 Plux+pT181 attenuator | 2 | 20 | 22 | -- |
9/5 Ptet+pT181 attenuator | 2 | 20 | 22 | -- |
9/5 Ptet+pT181 attenuator | 2 | 20 | 22 | -- |
9/5 Pconst+RBS-tetR+DT | 2 | 20 | 22 | -- |
9/16(2012) T7-His-FT | 2 | 20 | 22 | -- |
9/16(2012) pBr322 | 2 | 20 | 22 | -- |
Restriction Enzyme Digestion
9/5 Pbad/araC | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 4µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 18µL | 30µL |
9/5 RBS-luxI-DT | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 4µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 18µL | 30µL |
8/28 Plux | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 12µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 10µL | 30µL |
NC | 0.6µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |
9/5 Pconst(J23100) | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 5.7µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 16.3µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
9/5 Plac | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 6.0µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 16µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
8/31 Plux-RBS-GFP-DT-1 | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 9.4µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 12.6µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
8/20 Pconst-RBS-luxR-DT | E | S | X | P | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 4.5µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 13.5µL | 30µL |
NC | 0.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.8µL | 10µL |
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
9/5 RBS-luxI-DT | 7.6 | 1.56 | -1.42 |
9/5 Pbad/araC | 7.5 | 1.54 | 3.84 |
Electrophoresis
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 100bp ladder | -- | -- |
2 | aptamer 12_1R (pSB1C3)-1 | -- | -- |
3 | aptamer 12_1R (pSB1C3)-2 | -- | -- |
4 | pT181 antisense(pSB1C3)-1 | -- | -- |
5 | pT181 antisense(pSB1C3)-2 | -- | -- |
6 | pT181 antisense(pSB1C3)-3 | -- | -- |
7 | pT181 antisense(pSB1C3)-4 | -- | -- |
8 | 100bp ladder | -- | -- |
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 100bp ladder | -- | -- |
2 | aptamer 12_1R-DT-1 | -- | -- |
3 | aptamer 12_1R-DT-2 | -- | -- |
4 | aptamer 12_1R-DT-3 | -- | -- |
5 | aptamer 12_1R-DT-4 | -- | -- |
6 | pT181 attenuator(pSB1C3)-1 | -- | -- |
7 | pT181 attenuator(pSB1C3)-2 | -- | -- |
8 | RBS-lysis1-DT-1 | -- | -- |
9 | RBS-lysis1-DT-2 | -- | -- |
10 | 100bp ladder | -- | -- |
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 100bp ladder | -- | -- |
2 | RBS-lysis2-DT-1 | -- | -- |
3 | RBS-lysis2-DT-2 | -- | -- |
4 | RBS-lysis2-DT-3 | -- | -- |
5 | RBS-lysis2-DT-4 | -- | -- |
6 | RBS-lysis3-DT-1 | -- | -- |
7 | RBS-lysis3-DT-2 | -- | -- |
8 | 100bp ladder | -- | -- |
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 1kbp ladder | -- | -- |
2 | Pconst | SpeI | PstI |
3 | Pconst NC | -- | -- |
4 | Plac | SpeI | PstI |
5 | Plac NC | -- | -- |
6 | Plux | SpeI | PstI |
7 | Plux NC | -- | -- |
8 | Pconst-RBS-luxR-DT | EcoRI | XbaI |
9 | Pconst-RBS-luxR-DT NC | -- | -- |
10 | Plux-RBS-GFP-DT | EcoRI | SpeI |
11 | Plux-RBS-GFP-DT NC | -- | -- |
12 | 1kbp ladder | -- | -- |
5x M9 Medium (+EDTA)
volume | 10ml |
---|---|
Na2HPO4 | 60mg |
KH2PO4 | 30mg |
NaCl | 5mg |
NH4Cl | 10mg |
Fe(III)-EDTA | 1263.27mg |
MilliQ | up to 10 mL |
- autoclave at 121 °C for 20 min
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | |
2 | Pconst | SpeI & PstI |
3 | Pconst | SpeI & PstI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pconst | 5.3 | 1.98 | 0.06 |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | Plac | SpeI & PstI |
3 | Plac | SpeI & PstI |
5 | Plux | SpeI & PstI |
6 | Plux | SpeI & PstI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Plac (SpeI & PstI) | 5.2 | 1.82 | 0.36 |
Plux(SpeI & PstI) | 8.0 | 1.87 | 0.25 |
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | |
2 | Pconst-RBS-luxR-DT | EcoRI & XbaI |
3 | Pconst-RBS-luxR-DT | EcoRI & XbaI |
5 | Plux-RBS-GFP-DT | EcoRI & SpeI |
6 | Plux-RBS-GFP-DT | EcoRI & SpeI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pconst-RBS-luxR-DT(EcoRI & XbaI) | 30.6 | 1.84 | 1.16 |
Plux-RBS-GFP-DT (EcoRI & SpeI) | 21.8 | 1.87 | 0.98 |
DNA Purification
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
9/5 RBS-luxI-DT | 7.6 | 1.56 | -1.42 |
9/5 Pbad/araC | 7.5 | 1.54 | 3.84 |
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
8/9 J23100-5 | 243.0 | 1.90 | 1.81 |
Colony PCR
Sample | base pair |
---|---|
9/4 RBS-lysis3-DT-3 | 1210 |
9/4 RBS-lysis3-DT-4 | 1210 |
9/4 RBS-lysis3-DT-5 | 1210 |
9/4 RBS-lysis1-DT-3 | 613 |
9/4 Ptrc KaiC-1 | -- |
9/4 pSB4K5 | 1370 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 1min 25s | 30cycles |
Liquid Culture
Sample | medium |
---|---|
9/4 aptamer 12_1R-DT-1 | Plusgrow medium (+CP) |
9/4 pT181 attenuator(pSB1C3)-1 | Plusgrow medium (+CP) |
9/4 RBS-lysis2-DT-1 | Plusgrow medium (+CP) |
9/4 Ptrc-KaiC -1 | Plusgrow medium (+Amp) |
9/4 pSB4K5 -1 | Plusgrow medium (+Kan) |