From 2013.igem.org
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- | ===Miniprep===
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| ==Aug 20== | | ==Aug 20== |
Revision as of 11:10, 7 September 2013
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 | | | | |
|
| | | | |
|
Restriction Enzyme Digestion
じっけんしゃ
DNA | Buffer | restricion enzymeµL | restriction enzyme µL | MilliQ µL | total
|
(°°) | | | | |
|
Electrophoresis
じっけんしゃ
Lane | Sample | Enzyme1 | Enzyme2
|
(°Д°) | | |
|
Ligation
じっけんしゃ
Number | Vecter(concentration) | quantity µL | Inserter(concentration) | quantity µL
|
(>o<) | ( µg/mL) | | ( µg/mL) |
|
じっけんしゃ
Name | quantity | concentration[µg/mL] | 260/280 | 260/230
|
(´ω`) | | | |
|
(ΦωΦ^) | | | |
|
Colony PCR
No name
PreDenature | Denature | Annealing | Extension | cycle
|
°C | °C | °C | °C | -
|
(◎Д◎) | | | |
|
Liquid Culture
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230
|
(・▽・) | | |
|
Master Plate
Number | Use LB plate(+抗生物質)
|
ʅ(´◔౪◔)ʃ |
|
Aug 19
Colony PCR
Kojima
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
|
File:Igku Aug19electrophoresis2.png
LB Liquid Mideum Culture
Nakamoto
volume | 200ml
|
Bacto T2ypton | 2g
|
Bacto yeast extract | 1g
|
NaCl | 1g
|
Agar Pouder | 2g
|
Restriction Enzyme Digestion
Tatsui
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
|
Murata and Okazaki
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
|
Ligation
じっけんしゃ
Number | Vecter | quantity µL | Inserter | quantity µL
|
1 | RBS | 0.3 | lysis2 | 7.4
|
2(NC) | RBS | 0.3 | MilliQ | 7.4
|
Electrophoresis
じっけんしゃ
Lane | Sample | Enzyme1 | Enzyme2
|
1 | 100bp ladder | - | -
|
2 | 8/19 lysis1 | XbaI | PstI
|
3 | 8/19 lysis1 | XbaI | -
|
4 | 8/19 lysis1 | - | PstI
|
5 | 8/19 lysis1 | - | -
|
6 | 8/19 lysis1(Gel Extraction Product) | XbaI | PstI
|
Transformation
Murata and Kojima
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
|
じっけんしゃ
Lane | DNA | Enzyme
|
1 | 100bp ladder | -
|
2 | 8/19 lysis1① | XbaI, PstI
|
[fig before]
[fig after]
Name | concentration[µg/mL] | 260/280 | 260/230
|
8/19 lysis1① | 87.8 | 1.06 | 0.82
|
Liquid Culture
Ashida
Sample | medium
|
8/18 Ptet -1 | Plusgrow medium(+CP)
|
8/18 Ptet -2 | Plusgrow medium(+CP)
|
8/18 RBS-tetR-DT -1 | Plusgrow medium(+CP)
|
8/18 RBS-tetR-DT -2 | Plusgrow medium(+CP)
|
8/18 J23100-GFP -1 | Plusgrow medium(+Amp)
|
8/18 J23100-luxR -1 | Plusgrow medium(+Amp)
|
8/18 J23100-luxR -2 | Plusgrow medium(+Amp)
|
8/18 J23100-lacZα -1 | Plusgrow medium(+Amp)
|
8/18 RBS-lysis3 -1 | Plusgrow medium(+Amp)
|
8/18 RBS-lysis3 -2 | Plusgrow medium(+Amp)
|
Master Plate
Ashida
Number | Use LB plate(+Amp)
|
1 | 8/18 J23100-GFP -1
|
2 | 8/18 J23100-luxR -1
|
3 | 8/18 J23100-luxR -2
|
4 | 8/18 J23100+LacZα -1
|
5 | 8/18 RBS-lysis3 -1
|
6 | 8/18 RBS-lysis3 -2
|
Number | Use LB plate(+CP)
|
1 | 8/18 Ptet -1
|
1 | 8/18 Ptet -2
|
3 | 8/18 RBS-tetR-DT -1
|
4 | 8/18 RBS-tetR-DT -2
|
Aug 20
Miniprep
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
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 Digestion
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 XX°C for Xh
Transformation
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
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)
Tatsui
- 8/17 Plusgrow medium 50ml
- 7/22 5000x Ampicillin 10µl
- Measuring materials and putting them in a 50ml tube
Liquid culture
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 Digestion
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
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 (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
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 |
|
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
No name
DNA | Buffer | restricion enzymeµL | restriction enzyme µL | MilliQ µL | total
|
(;ω;) | | | | |
|
Restriction Enzyme Digestion
No name
(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 | 1µL | 0.1µL | 8.6µL | 10µL
|
Aug 24
Transformation
No 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 23==