Template:Kyoto/Notebook/Sep 23
From 2013.igem.org
Contents |
Sep 23
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | |
3-4 | Pcon-RBS-tetR-PT |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pcon-PT181 anti E+S | 46.7 | 1.06 | 0.93 |
Pcon-RBS-tetR-DT E+X | 119.3 | 0.99 | |
RBS-8FP-DT E+X | 87.7 | 1.00 | 0.98 |
ap42_1R-DT E+X | 68.8 | 1.32 | 1.03 |
Pcon-RBS-tetR-DT 1-2 A | 203.0 | 1.66 | 1.79 |
Pcon-RBS-tetR-DT 3-2 A | 67.7 | 2.64 | 1.28 |
Pcon-RBS-GFP-DT 1-S A | 185.6 | 1.88 | 2.04 |
Pcon-RBS-GFP-DT 2-S A | 298.6 | 1.68 | 1.55 |
Pcon-tetR-DT E-2 A | 202.4 | 1.57 | 1.66 |
PCR
genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA_fwd primer | SasA_rev primer | MilliQ | total |
---|---|---|---|---|---|---|---|---|
9/8 Pcon-pT181 anti | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
8/20 Ptet(1) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
9/16 Plac 2 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94 °C | 98 °C | 57 °C | 68 °C | -- |
2min | 10sec | 30sec | 20sec | 30cycles |
Ligation
state | Vector | Inserter | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | 9/21 Pcon-RBS-GFP-DT E+X | 47.5 µg/mL | 9/15 Pcon-RBS-tetR-DT E+S | 59 µg/mL | 2.3 µg/mL |
experiment | 9/21 Pcon-RBS-GFP-DT E+X | 47.5 µg/mL | 9/15 Pcon-apt12_1R-DT E+S | 17 µg/mL | 2.4 µg/mL |
experiment | 9/21 Pcon-RBS-GFRDT E+X | 2.1 µg/mL | 9/15 Pcon-DT181 atte-DT | 21 µg/mL | 3.0 µg/mL |
experiment | 9/22 apt 12_1R-DT E+X | 67.8 µg/mL | 9/18 Pcon-DT181 atte E+S | 5.7 µg/mL | 3.5 µg/mL |
experiment | 9/22RBS-GFP-DT E+X | 87.7 µg/mL | 9/18 Pcon-DT18 atte E+S | 6.7 µg/mL | 3.5 µg/mL |
experiment | 9/13 DT E+X | 25.4 µg/mL | 9/22 Pcon-DT181 anti E+S | 46.8 µg/mL | 2.3 µg/mL |
PCR
genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA_fwd primer | SasA_rev primer | MilliQ | total |
---|---|---|---|---|---|---|---|---|
Pcon-atte-DT (E-1A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
Pcon-atte-DT (2-1A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
Pcon-anti-spin-DT (1-2A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
Pcon-anti-spin-DT (E-1A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94 °C | 98 °C | 57 °C | 68 °C | -- |
2min | 10s | 30s | 45s | 30cycle |
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
9/22 Plac-DT181 atte-1 | 204.0 | 2.00 | 1.95 |
9/22 Plac-DT181 atte-2 | 195.3 | 1.84 | 1.62 |
9/22 Plac-DT181 anti-1 | 188.2 | 1.87 | 2.15 |
9/22 Plac-DT181 anti-2 | 165.0 | 1.95 | 2.66 |
Electrophoresis
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1,6 | 100bp ladder(Promega) | ||
2 | Pcon-atte-DT (E-1A) | ||
3 | Pcon-atte-DT (2-1A) | ||
4 | Pcon-anti-spi-DT (1-2A) | ||
5 | Pcon-anti-spi-DT (E-1A) |
Gel Extraction
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pcon atte-DT(E-1A) | 166.7 | 1.51 | 0.99 |
Pcon atte-DT(2-1A) | 122.1 | 1.75 | 1.20 |
Pcon anti spi DT(1-2A) | 99.1 | 1.86 | 1.81 |
Electrophoresis
Transformation
Name | Sample | Competent Cells | Total | Plate |
---|---|---|---|---|
pSB1C3 | 2µL | 20µL | 22µL |
Ligation
state | Vector | Inserter | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | 9/19 Plac S+P | 24.6 µg/mL | 9/18 ape12_1R-DT X+P | 4.5 µg/mL | 2 µg/mL |
experiment | 9/21 RBS-GFP-DT E+X | 88.4 µg/mL | 9/15 Pcon-pT181 atte E+S | 21 µg/mL | 2 µg/mL |
experiment | 9/13 PSB1C3 X+P | 26.0 µg/mL | 9/16 pT181 atte X+P | 8.2 µg/mL | 2 µg/mL |
Colony PCR
Sample | base pair |
---|---|
9/22 Pcon-PT181 attenuator12-1R-DT(1~8) | -- |
9/22 Pcon-PT181 attenuator(1~4) | -- |
9/20 Plac+aptamer12-1R-DT(1~4) | -- |
Colony PCR
Sample | base pair |
---|---|
9/20 Plac-PT181 attenuator(1~4) | 664 |
9/20 Plac-PT181 antisense(1~6) | 369 |
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6) | 859 |
9/20 Pcon-PT181 antisense-spinach-DT(1~8) | 723 |