From 2013.igem.org
template
transformation
じっけんしゃ
| Name | Well | Sample | Competent Cells | Total | Plate
|
| いでんし | ぱーつのうぇる | なんちゃらµL | ごにょごにょµL | ほにゃららµL | こーせーぶっしつ
|
| いかどうよう | | | | |
|
| | | | | |
|
Aug 20
Miniprep
by kojima
| DNA | concentration[µg/µL] | 260/280 | 260/230
|
| 8/18 Ptet -1 | 220 | 1.69 | 1.87
|
| 8/18 Ptet -2 | 210 | 1.69 | 1.71
|
| 8/18 RBS-tetR-DT -1 | 236 | 1.63 | 1.69
|
| 8/18 RBS-tetR-DT -2 | 182 | 1.65 | 1.98
|
| 8/18 J23100-RBS-GFP-DT -1 | 334 | 1.71 | 2.12
|
| 8/18 J23100-RBS-luxR-DT -1 | 440 | 1.67 | 1.60
|
| 8/18 J23100-RBS-luxR-DT -2 | 344 | 1.68 | 1.83
|
| 8/18 J23100-RBS-lacZα-DT -1 | 280 | 1.70 | 1.81
|
| 8/18 RBS-lysis3 -1 | 282 | 1.67 | 1.76
|
| 8/18 RBS-lysis3 -2 | 212 | 1.30 | 1.35
|
LB Culture Medium
by Okazaki
| volume | 200mL
|
| Bacto Trypton | 2g
|
| Bacto yeast extract | 1g
|
| Nacl | 1g
|
| Agar Powder | 2g
|
| Amp | 40µl
|
1.Measuring reagents and put they in the Erlenmeyer flask
2.diluting in measuring cylinder to 200ml total
3.autoclaving
4.adding ampicirine 40ml
not written yet
Ristriction Enzyme Proccessing
by Kojima and Ashida
| 8/20 Ptet -1(220µg/mL) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total
|
| 4.5 | 1 | 1 | 3 | 3 | 17.5 | 30
|
| 0.5 | 0.2 | 0 | 1 | 1 | 7.3 | 10
|
| 0.5 | 0 | 0.2 | 1 | 1 | 7.3 | 10
|
| 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10
|
| 8/20 RBS-tetR-DT -2(182µg/mL) | EcoRI | SpeI | 10x buffer H | MilliQ | total
|
| 5.5 | 1 | 1 | 3 | 19.5 | 30
|
| 0.6 | 0.2 | 0 | 1 | 8.2 | 10
|
| 0.6 | 0 | 0.2 | 1 | 8.2 | 10
|
| 0.6 | 0 | 0 | 1 | 8.4 | 10
|
| 8/20 J23100-RBS-GFP-DT(334µg/ml) | EcoRI | SpeI | 10x buffer H | MilliQ | total
|
| 3.0 | 1 | 1 | 3 | 22 | 30
|
| 0.3 | 0.2 | 0 | 1 | 8.5 | 10
|
| 0.3 | 0 | 0.2 | 1 | 8.5 | 10
|
| 0.3 | 0 | 0 | 1 | 8.7 | 10
|
| 8/20 J23100-RBS-luxR-DT -2(344µg/mL) | EcoRI | XbaI | BSA | 10x buffer M | MilliQ | total
|
| 2.9 | 1 | 1 | 3 | 3 | 19.1 | 30
|
| 0.3 | 0.2 | 0 | 1 | 1 | 7.5 | 10
|
| 0.3 | 0 | 0.2 | 1 | 1 | 7.5 | 10
|
| 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10
|
| 8/20 RBS-lysis3 -1(282µg/mL) | EcoRI | SpeI | 10x buffer H | MilliQ | total
|
| 3.5 | 1 | 1 | 3 | 21.5 | 30
|
| 0.4 | 0.2 | 0 | 1 | 8.4 | 10
|
| 0.4 | 0 | 0.2 | 1 | 8.4 | 10
|
| 0.4 | 0 | 0 | 1 | 8.6 | 10
|
| 8/17 DT -1(188µg/mL) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total
|
| 5.3 | 1 | 1 | 3 | 3 | 16.7 | 30
|
| 0.3 | 0.2 | 0 | 1 | 1 | 7.5 | 10
|
| 0.3 | 0 | 0.2 | 1 | 1 | 7.5 | 10
|
| 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10
|
incubate ~20:00
Transformation
by Nakamoto
| Name | Sample | Competent Cells(XL10-gold) | Total | Plate
|
| tRNA-Spinach-tRNA | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| tetR-aptamer 12_PC(076) | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| tetR-aptamer 12_1R(113) | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| tetR-aptamer 12_1M(105) | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| pT181 attenuator | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| Fusin1 attenuator | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| Fusion3m2 attenuator | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| pT181 antisense | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| Fusion1 antisense | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
| Fusion6 antisense | 1µL | 10µL | 11µL | 8/19 LB+Amp
|
incubate 37°C overnight 20:34~
Electrophoresis
by Kojima
| Lane | Sample | Enzyme1 | Enzyme2
|
| 1 | 8/20 Ptet -1 | EcoRI | XbaI
|
| 2 | 8/20 Ptet -1 | EcoRI | -
|
| 3 | 8/20 Ptet -1 | - | XbaI
|
| 4 | 8/20 Ptet -1 | - | -
|
| 5 | 8/20 RBS-tetR-DT -2 | EcoRI | SpeI
|
| 6 | 8/20 RBS-tetR-DT -2 | EcoRI | -
|
| 7 | 8/20 RBS-tetR-DT -2 | - | SpeI
|
| 8 | 8/20 RBS-tetR-DT -2 | - | -
|
| 9 | 1kbp ladder | - | -
|
| 10 | 8/20 J23100-RBS-GFP-DT -1 | EcoRI | SpeI
|
| 11 | 8/20 J23100-RBS-GFP-DT -1 | EcoRI | -
|
| 12 | 8/20 J23100-RBS-GFP-DT -1 | - | SpeI
|
| 13 | 8/20 J23100-RBS-GFP-DT -1 | - | -
|
| 14 | 8/20 J23100-RBS-luxR-DT -1 | EcoRI | XbaI
|
| 15 | 8/20 J23100-RBS-luxR-DT -1 | EcoRI | -
|
| 16 | 8/20 J23100-RBS-luxR-DT -1 | - | XbaI
|
| 17 | 8/20 J23100-RBS-luxR-DT -1 | - | -
|
| 18 | 1kbp ladder | - | -
|
| 19 | 8/20 RBS-lysis3 -1 | EcoRI | SpeI
|
| 20 | 8/20 RBS-lysis3 -1 | EcoRI | -
|
| 21 | 8/20 RBS-lysis3 -1 | - | SpeI
|
| 22 | 8/20 RBS-lysis3 -1 | - | -
|
| 23 | 1kbp ladder | - | -
|
| 24 | 8/17 DT | EcoRI | XbaI
|
| 25 | 8/17 DT | EcoRI | -
|
| 26 | 8/17 DT | - | XbaI
|
| 27 | 8/17 DT | - | -
|
Aug 21
Plusgrow medium(+amp)
by Tatsui
- 8/17 Plusgrow medium 50ml
- 7/22 5000× Ampicillin 10µl
- Measuring materials and putting them in a 50ml tube
Liquid culture
by tatsui
| Sample | medium
|
| 8/20 tRNA-spinach | 8/21 Plusgrow medium(+amp)
|
| 8/20 TetR-aptamer 12_P(1076) | 8/21 Plusgrow medium(+amp)
|
| 8/20 TetR-aptamer 12_1R(113) | 8/21 Plusgrow medium(+amp)
|
| 8/20 TetR-aptamer 12_1M(105) | 8/21 Plusgrow medium(+amp)
|
| 8/20 PT181 attenuator | 8/21 Plusgrow medium(+amp)
|
| 8/20 Fusion1 attenuator | 8/21 Plusgrow medium(+amp)
|
| 8/20 Fusion3m2 attenuator | 8/21 Plusgrow medium(+amp)
|
| 8/20 PT181 antisense | 8/21 Plusgrow medium(+amp)
|
| 8/20 Fusion1 antisense | 8/21 Plusgrow medium(+amp)
|
| 8/20 Fusion6 antisense | 8/21 Plusgrow medium(+amp)
|
incubate 37°C 10hour
Ristriction Enzyme Proccessing
by Kojima and Nakamoto
| 8/20 Ptet-(1) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total
|
| 4.5 | 1 | 1 | 3 | 3 | 17.5 | 30
|
| 0.5 | 0.2 | 0 | 1 | 1 | 7.3 | 10
|
| 0.5 | 0 | 0.2 | 1 | 1 | 7.3 | 10
|
| 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10
|
| 8/20 Pconst-RBS-luxR-DT-(2) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total
|
| 2.9 | 1 | 1 | 3 | 3 | 19.1 | 30
|
| 0.3 | 0.2 | 0 | 1 | 1 | 7.5 | 10
|
| 0.3 | 0 | 0.2 | 1 | 1 | 7.5 | 10
|
| 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10
|
| 8/17 DT | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total
|
| 5.3 | 1 | 1 | 3 | 3 | 16.7 | 30
|
| 0.5 | 0.2 | 0 | 1 | 1 | 7.3 | 10
|
| 0.5 | 0 | 0.2 | 1 | 1 | 7.3 | 10
|
| 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10
|
incubate 37°C 1h
Electrophoresis
Aug 22
Liquid culture
by Nakamoto
| Sample | Medium
|
| 8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Pcon-RBS-GFP-DT control (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Pcon-RBS-GFP-DT control (2) | 8/21 Plusgrow medium(+amp)
|
| 8/21 RBS-lysis1 (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 RBS-lysis1 (2) | 8/21 Plusgrow medium(+amp)
|
| 8/21 RBS control (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 RBS control (2) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Ptet(pm)-RBS-tetR-DT (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Ptet(pm)-RBS-tetR-DT (2) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Ptet(pm) control (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 Ptet(pm) control | 8/21 Plusgrow medium(+amp)
|
| 8/21 Plux-RBS-GFP-DT (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 pSB1C3(BBa_J04450) (1) | 8/21 Plusgrow medium(+amp)
|
| 8/21 pSB1C3(BBa_J04450) (2) | 8/21 Plusgrow medium(+amp)
|
incubate 37℃ 10hour
colony PCR
by Kojima
| Sample | base pair
|
| 8/21 RBS-lysis1(1) | 400
|
| 8/21 RBS-lysis1(2) | 400
|
| 8/21 RBS control(1) |
|
| 8/21 RBS control(2) |
|
| negative control |
|
| Sample | base pair
|
| 8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1) | 2143
|
| 8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2) | 2143
|
| 8/21 Pcon-RBS-luxR control(1) |
|
| 8/21 Pcon-RBS-luxR control(2) |
|
| negative control
|
| Sample | base pair
|
| 8/21 Ptet(pm)-RBS-tetR-DT (1) |
|
| 8/21 Ptet(pm)-RBS-tetR-DT (2) |
|
| 8/21 Ptet(pm) control (1) |
|
| 8/21 Ptet(pm) control (2) |
|
| 8/21 Plux-RBS-GFP-DT (1) |
|
| 8/21 pSB1C3(BBa_J04450) (1) |
|
| 8/21 pSB1C3(BBa_J04450) (2) |
|
| negative control |
|
"
