Team:Kyoto/Notebook
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Contents |
template
Transformation
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 | |||||
Restriction Enzyme Digestion
DNA | Buffer | restricion enzymeµL | restriction enzyme µL | MilliQ µL | total |
---|---|---|---|---|---|
(°°) |
Electrophoresis
DNA | Buffer | restricion enzymeµL | restriction enzyme µL | MilliQ µL | total |
---|---|---|---|---|---|
(°Д°) |
Ligation
Vecter(name & concentration) | quantity µL |
---|---|
(>o<) |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
(>ω・)b | ||
Name | quantity | concentration | 280/260 | 260/230 |
---|---|---|---|---|
(´ω`) | ||||
(ΦωΦ^) |
Colony PCR
Sample | base pair |
---|---|
(`・ω・´) |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
°C | °C | °C | °C | - |
(◎Д◎) |
Liquid Culture
Sample | medium |
---|---|
(・ω・) | |
(・X・) |
Miniprep
DNA | concentration | µg/µL | 260/280 | 260/230 |
---|---|---|---|---|
(・▽・) |
Master Plate
- material
Aug 19
Colony PCR
numbar | Sample | base pair |
---|---|---|
1 | 8/18 J23100 control -(1) | 1142 |
2 | 8/18 J23100-RBS-GFP-DT -(1) | 1156 |
3 | 8/18 J23100-RBS-GFP-DT -(2) | 1156 |
4 | 8/18 J23100-RBS-luxR-DT -(1) | 1271 |
5 | 8/18 J23100-RBS-luxR-DT -(2) | 1271 |
6 | 8/18 J23100-RBS-lacZα-DT -(1) | 670 |
7 | 8/18 J23100-RBS-lacZα-DT -(2) | 670 |
8 | 8/18 RBS-lysis3 -(1) | 997 |
9 | 8/18 RBS-lysis3 -(2) | 997 |
10 | 8/18 P0440(RBS-tetR-DT) -(1) | 1154 |
11 | 8/18 P0440(RBS-tetR-DT) -(2) | 1154 |
12 | negative control | - |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | - |
5min | 30s | 30s | 1min | 30cycles |
File:Igku Aug19electrophoresis1.png
numbar | Sample | base pair |
---|---|---|
17 | 8/18 RBS control -(1) | - |
18 | 8/18 R0040(Ptet) -(1) | 686 |
19 | 8/18 R0040(Ptet) -(2) | 686 |
20 | negative control | - |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | - |
5min | 30s | 30s | 30s | 30cycles |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | - |
3 | 8/18 K117000(lysis1) -1 | XbaI & PstI |
5 | 8/18 K117000(lysis2) -1 | XbaI & PstI |
File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4
LB Liquid Mideum Culture
volume | 200ml |
---|---|
Bacto T2ypton | 2g |
Bacto yeast extract | 1g |
NaCl | 1g |
Agar Pouder | 2g |
Restriction Enzyme Digestion
lysis2-1 | XbaⅠ | PstⅠ | BSA | 10xbuffer | MilliQ | total |
---|---|---|---|---|---|---|
10µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
2µL | 0.2µL | -- | 1µL | 1µL | 5.8µL | 10µL |
2µL | -- | 0.2µL | 1µL | 1µL | 5.8µL | 10µL |
2µLL | -- | -- | 1µL | 1µ | 6µL | 10µL |
- at 37°C, for 1h
8/19 lysis1-1 | 10xbuffer | XbaⅠ | PstⅠ | BSA | MilliQ | total |
---|---|---|---|---|---|---|
2µL | 2µL | 0.5µL | 0.8µL | 2µL | 13µL | 20µL |
0.5µL | 1µL | 0.2µL | -- | 1µL | 7.3µL | 10µL |
0.5µL | 1µL | -- | 0.2µL | 1µL | 7.3µL | 10µL |
0.5µL | 1µL | -- | -- | 1µL | 7.5µL | 10µL |
- at 37℃, for 1h
Transformation
Name | Well | Sample | Competent Cells | Total | Plate |
---|---|---|---|---|---|
8/18 Plux-RBS-GFP-DT | ぱーつのうぇる | 2µL | 20µL | ほにゃららµL | CP |
8/18 Plux(NC) | 2µL | 20µL | µL | CP | |
8/19 RBS+lysis2 | 2µL | 20µL | µL | Amp | |
8/15 Pbad-araC | 2µL | 20µL | µL | Kana |
- Incubate at 17°C
Gel Extraction
Master Plate
Use LB plate(+Amp) |
---|
8/18 J23100-6pp(Amp) |
8/18 J23100-luxR(Amp) |
8/18 J23100-luxR(Amp) |
8/18 J23100+LacZα(Amp) |
8/18 RBS-lysis3(Amp) |
8/18 RBS-lysis3(Amp) |
Use LB plate(+CP) |
---|
8/18 Ptet |
8/18 RBS-tetR-DT |
8/18 RBS-tetR-DT |
Miniprep
Aug 20
Miniprep
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
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 Digestion
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 XX°C for Xh
Transformation
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
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)
- material
- 8/17 Plusgrow medium 50ml
- 7/22 5000x Ampicillin 10µl
- Measuring materials and putting them in a 50ml tube
Liquid culture
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 Digestion
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
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 (2) | 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°C 10hour
Colony PCR
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 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | - |
5min | 30s | 30s | 30s | 30cycles |
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 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | - |
5min | 30s | 30s | 30s | 30cycles |
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 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | - |
5min | 30s | 30s | 30s | 30cycles |
Aug 23
Electrophoresis
DNA | Buffer | restricion enzymeµL | restriction enzyme µL | MilliQ µL | total |
---|---|---|---|---|---|
(;ω;) |
Restriction Enzyme Digestion
操作 | TecR aptamer | EcoRⅠ | SpeⅠ | 10xBuffer B | 100xBSA | MilliQ | total |
---|---|---|---|---|---|---|---|
E+S | 2.7µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |
操作 | TecRaptamer12-1RQ1 | EcoRⅠ | SpeⅠ | Buffer B | BSA | MilliQ | total |
---|---|---|---|---|---|---|---|
E+S | 2.6µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23.1µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 1µL | 1µL | 8.6µL | 10µL |
操作 | TetR aptamer12.1M(1) | EcoRⅠ | SpeⅠ | Buffer B | BSA | MilliQ | total | |||||||||||||||||||||
E+S | 2.7µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23µL | 30µL | |||||||||||||||||||||
NC | 0.3µL | 0µL | 0µL |
templateTransformation
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