Team:Kyoto/Notebook
From 2013.igem.org
(Difference between revisions)
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{| class="wikitable" | {| class="wikitable" | ||
- | !Name|| ||NC | + | !Name||-?-||NC |
|- | |- | ||
- | |8/21 Pcn-RBS-luxR-DT||2.3||2.3 | + | |8/21 Pcn-RBS-luxR-DT(+Amp)||2.3||2.3 |
|- | |- | ||
|8/21 Pcn-RBS-GFP-DT||1.7||0 | |8/21 Pcn-RBS-GFP-DT||1.7||0 | ||
Line 907: | Line 907: | ||
{| class="wikitable" | {| class="wikitable" | ||
- | !Name|| ||NC | + | !Name||-?-||NC |
|- | |- | ||
- | |8/18 Plux||1.1||1.1 | + | |8/18 Plux(+CP)||1.1||1.1 |
|- | |- | ||
|8/19 RBS-GFP-DT||5||0 | |8/19 RBS-GFP-DT||5||0 | ||
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|} | |} | ||
+ | {| class="wikitable" | ||
+ | !Name||-?-||NC | ||
+ | |- | ||
+ | |8/18 RBS(+Amp)||2.2||-- | ||
+ | |- | ||
+ | |8/19 lysis1||6.0||-- | ||
+ | |- | ||
+ | |Ligation High buffer||4.1||-- | ||
+ | |- | ||
+ | |MilliQ||0||-- | ||
+ | |- | ||
+ | |total||12.3||-- | ||
+ | |} | ||
+ | |||
+ | {| class="wikitable" | ||
+ | !Name||-?-||NC | ||
+ | |- | ||
+ | |8/18 RBS(+Amp)||2.2||2.2 | ||
+ | |- | ||
+ | |8/19 lysis2||3.6||0 | ||
+ | |- | ||
+ | |Ligation High buffer||2.9||2.9 | ||
+ | |- | ||
+ | |MilliQ||0||3.6 | ||
+ | |- | ||
+ | |total||8.7||8.7 | ||
+ | |} | ||
</div> | </div> |
Revision as of 06:55, 9 September 2013
Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Material and method | Safety | Attributions |
---|
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 enzyme | restriction enzyme | MilliQ | total |
---|---|---|---|---|---|
(°°) µL | µL | µL | µL | µL | µL |
Electrophoresis
Ligation
Number | Vecter(concentration) | quantity µL | Inserter(concentration) | quantity µL |
---|---|---|---|---|
(>o<) | ( µg/mL) | ( µg/mL) |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
(>ω・)b | ||
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
Name | quantity | concentration[µg/mL] | 260/280 | 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/mL] | 260/280 | 260/230 |
---|---|---|---|
(・▽・) |
Master Plate
Number | Use LB plate (+抗生物質) |
---|---|
ʅ(´◔౪◔)ʃ |
Aug 19
Colony PCR
Sample | base pair |
---|---|
8/18 J23100 control -(1) | 1142 |
8/18 J23100-RBS-GFP-DT -(1) | 1156 |
8/18 J23100-RBS-GFP-DT -(2) | 1156 |
8/18 J23100-RBS-luxR-DT -(1) | 1271 |
8/18 J23100-RBS-luxR-DT -(2) | 1271 |
8/18 J23100-RBS-lacZα-DT -(1) | 670 |
8/18 J23100-RBS-lacZα-DT -(2) | 670 |
8/18 RBS-lysis3 -(1) | 997 |
8/18 RBS-lysis3 -(2) | 997 |
8/18 P0440(RBS-tetR-DT) -(1) | 1154 |
8/18 P0440(RBS-tetR-DT) -(2) | 1154 |
negative control | -- |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 1min | 30cycles |
File:Igku Aug19electrophoresis1.png
Sample | base pair |
---|---|
8/18 RBS control -(1) | -- |
8/18 R0040(Ptet) -(1) | 686 |
8/18 R0040(Ptet) -(2) | 686 |
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(lysis2) -1 | XbaI & PstI |
5 | 8/18 K117000(lysis2) -1 | XbaI & PstI |
File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
8/18 lysis2-1(XbaI & PstI) | 3.1 | 3.64 | 0.19 |
8/18 lysis2-1(XbaI & PstI) | 3.4 | 1.89 | 0.13 |
LB Medium Plate
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°C, for 1h
Ligation
Number | Vecter | quantity µL | Inserter | quantity µL |
---|---|---|---|---|
1 | RBS | 0.3 | lysis2 | 7.4 |
2(NC) | RBS | 0.3 | MilliQ | 7.4 |
Electrophoresis
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
Name | Well | Sample | Competent Cells | Plate |
---|---|---|---|---|
8/18 Plux+RBS-GFP-DT | -- | 2µL | 20µL | CP |
8/18 Plux(NC) | -- | 2µL | 20µL | CP |
8/19 RBS+lysis2 | -- | 2µL | 20µL | Amp |
8/15 Pbad-araC | Plate5-14N | 2µL | 20µL | Kan |
- Incubate at 37°C
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
2 | 8/19 lysis1 ① | XbaI & PstI |
[fig before] [fig after]
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
8/19 lysis1 ①(XbaI & PstI) | 87.8 | 1.06 | 0.82 |
Liquid Culture
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
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
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 Medium Plate
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.
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 |
Transformation
Name | Sample | Competent Cells(XL10-gold) | Total | Plate |
---|---|---|---|---|
tRNA-Spinach-tRNA | 1µL | 10µL | 11µL | Amp |
tetR-aptamer 12_PC(076) | 1µL | 10µL | 11µL | Amp |
tetR-aptamer 12_1R(113) | 1µL | 10µL | 11µL | Amp |
tetR-aptamer 12_1M(105) | 1µL | 10µL | 11µL | Amp |
pT181 attenuator | 1µL | 10µL | 11µL | Amp |
Fusin1 attenuator | 1µL | 10µL | 11µL | Amp |
Fusion3m2 attenuator | 1µL | 10µL | 11µL | Amp |
pT181 antisense | 1µL | 10µL | 11µL | Amp |
Fusion1 antisense | 1µL | 10µL | 11µL | Amp |
Fusion6 antisense | 1µL | 10µL | 11µL | 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
- 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 -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 tRNA-spinach -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 TetR-aptamer 12_P(1076) -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 TetR-aptamer 12_P(1076) -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 TetR-aptamer 12_1R(113) -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 TetR-aptamer 12_1R(113) -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 TetR-aptamer 12_1M(105) -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 TetR-aptamer 12_1M(105) -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 PT181 attenuator -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 PT181 attenuator -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion1 attenuator -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion1 attenuator -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion3m2 attenuator -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion3m2 attenuator -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 PT181 antisense -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 PT181 antisense -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion1 antisense -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion1 antisense -(2) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion6 antisense -(1) | 8/21 Plusgrow medium(+Amp) |
8/20 Fusion6 antisense -(2) | 8/21 Plusgrow medium(+Amp) |
- incubate 37°C 10hour
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | -- |
2 | 8/20 Ptet -(1) | EcoRI & XbaI |
3 | ||
5 | 8/20 RBS-tetR-DT -(2) | EcoRI & SpeI |
6 | ||
8 | 8/20 Pconst-RBS-GFP-DT -(1) | EcoRI & SpeI |
9 | ||
11 | 8/20 RBS-lysis3 -(1) | EcoRI & SpeI |
12 |
[fig bifore][fig after]
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Ptet (EcoRI & XbaI) | 21 | 1.55 | 0.78 |
RBS-tetR-DT (EcoRI & SpeI) | 12 | 1.45 | 0.62 |
Pconst-RBS-GFP-DT (EcoRI & SpeI) | 11 | 1.36 | 0.60 |
RBS-lysis3 (EcoRI & SpeI) | 10 | 1.26 | 0.50 |
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
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 1kbp ladder | -- | -- |
2 | 8/20 Ptet -(1) | EcoRI | XbaI |
3 | 8/20 Ptet -(1) | EcoRI | -- |
4 | 8/20 Ptet -(1) | -- | XbaI |
5 | 8/20 Ptet -(1) | -- | -- |
6 | 8/20 J23100-RBS-luxR-DT -(2) | EcoRI | XbaI |
7 | 8/20 J23100-RBS-luxR-DT -(2) | EcoRI | -- |
8 | 8/20 J23100-RBS-luxR-DT -(2) | -- | XbaI |
9 | 8/20 J23100-RBS-luxR-DT -(2) | -- | -- |
10 | 8/20 DT | EcoRI | XbaI |
11 | 8/20 DT | EcoRI | -- |
12 | 8/20 DT | -- | XbaI |
13 | 8/20 DT | -- | -- |
14 | 1kbp ladder | -- | -- |
15 | 8/20 Ptet -(1) | EcoRI | XbaI |
- The reason why lane 2 and 15 were same is that the well of lane 2 might be broken.
[fig]
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 1kbp ladder | -- | -- |
2 | 8/20 J23100-RBS-luxR-DT -(2) | EcoRI | XbaI |
3 | 8/20 DT | EcoRI | XbaI |
[fig]
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kb ladder | -- |
2 | 8/21 J23100-RBS-luxR-DT 25µL | E+X |
3 | 8/21 J23100-RBS-luxR-DT 25µL | E+X |
4 | -- | -- |
5 | 8/21 Ptet 25µL | E+X |
6 | 8/21 Ptet 25µL | E+X |
Electrophoresis
Lane | Sample | EcoRI | XbaI |
---|---|---|---|
1 | 1kb ladder | -- | -- |
2 | 8/21 DT | + | + |
3 | 8/21 DT | + | -- |
4 | 8/21 DT | -- | + |
5 | 8/21 DT | -- | -- |
Ligation
Number | Vecter(concentration) | quantity µL | Inserter(concentration) | quantity µL |
---|---|---|---|---|
1 | 8/21 Pcon-RBS-luxR-DT(E+X) (11ng/µLL) | 2.3µL | 8/21 Pcon-RBS-GFP-DT(E+S)(15ng/µLL) | 1.7µL |
2 | 6/18 Plux(S+P) (22.1ng/µLL) | 1.1µL | 8/19 RBS-GFP-DT(E+S) (4.8ng/µLL) | 5µL |
3 | 8/18 RBS(S+P) (11.2µg/mL) | 2.2µL | 8/19 lysis1(X+P) (4.2µg/µL) | 6.0µL |
4 | 8/18 RBS(S+P) (11.2µg/mL) | 2.2µL | 8/19 lysis2(X+P) (7.0ng/µL) | 3.6µL |
5 | 8/21 Ptec pm(E+X) (17µg/mL) | 1.5µL | 8/20 RBS-tetR-DT(2)(E+S) (12ng/µL) | 2.1µL |
Name | -?- | NC |
---|---|---|
8/21 Pcn-RBS-luxR-DT(+Amp) | 2.3 | 2.3 |
8/21 Pcn-RBS-GFP-DT | 1.7 | 0 |
Ligation High buffer | 2 | 2 |
MilliQ | 0 | 1.7 |
total | 6 | 6 |
Name | -?- | NC |
---|---|---|
8/18 Plux(+CP) | 1.1 | 1.1 |
8/19 RBS-GFP-DT | 5 | 0 |
Ligation High buffer | 3.1 | 3.1 |
MilliQ | 0 | 5 |
total | 9.2 | 9.2 |
Name | -?- | NC |
---|---|---|
8/18 RBS(+Amp) | 2.2 | -- |
8/19 lysis1 | 6.0 | -- |
Ligation High buffer | 4.1 | -- |
MilliQ | 0 | -- |
total | 12.3 | -- |
Name | -?- | NC |
---|---|---|
8/18 RBS(+Amp) | 2.2 | 2.2 |
8/19 lysis2 | 3.6 | 0 |
Ligation High buffer | 2.9 | 2.9 |
MilliQ | 0 | 3.6 |
total | 8.7 | 8.7 |
Transformation
LB Medium Plate
Gel Extraction
Miniprep
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 | 2min | 30cycles |
Sample | base pair |
---|---|
8/21 Ptet-RBS-tetR-DT (1) | 1216 |
8/21 Ptet-RBS-tetR-DT (2) | 1216 |
8/21 Ptet control (1) | -- |
8/21 Ptet control (2) | -- |
8/21 Plux-RBS-GFP-DT (1) | 1227 |
8/21 pSB1C3(BBa_J04450) (1) | 1353 |
8/21 pSB1C3(BBa_J04450) (2) | 1353 |
negative control | -- |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | -- |
5min | 30s | 30s | 1min | 30cycles |
Electrophoresis
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | pT181antisense | EcoRI | SpeI |
2 | pT181antisense | EcoRI | -- |
3 | pT181antisense | -- | SpeI |
4 | pT181antisense | -- | -- |
5 | pT181antisense | XbaI | PstI |
6 | pT181antisense | XbaI | -- |
7 | pT181antisense | -- | PstI |
8 | pT181antisense | -- | -- |
9 | 100bp ladder | -- | -- |
10 | Fusion1 attenuator | EcoRI | SpeI |
11 | Fusion1 attenuator | EcoRI | -- |
12 | Fusion1 attenuator | -- | SpeI |
13 | Fusion1 attenuator | -- | -- |
14 | Fusion1 attenuator | XbaI | PstI |
15 | Fusion1 attenuator | XbaI | -- |
16 | fusion1 attenuator | -- | PstI |
17 | fusion1 attenuator | -- | -- |
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | Fusion3 3m2 attenuator | EcoRI | SpeI |
2 | Fusion3 3m2 attenuator | EcoRI | -- |
3 | Fusion3 3m2 attenuator | -- | SpeI |
4 | Fusion3 3m2 attenuator | -- | -- |
5 | Fusion3 3m2 attenuator | XbaI | PstI |
6 | Fusion3 3m2 attenuator | XbaI | -- |
7 | Fusion3 3m2 attenuator | -- | PstI |
8 | Fusion3 3m2 attenuator | -- | -- |
9 | 100bp ladder | -- | -- |
10 | aptamer 12-1M | EcoRI | SpeI |
11 | aptamer 12-1M | EcoRI | -- |
12 | aptamer 12-1M | -- | SpeI |
13 | aptamer 12-1M | -- | -- |
14 | aptamer 12-P | EcoRI | SpeI |
15 | aptamer 12-P | EcoRI | -- |
16 | aptamer 12-1P | -- | SpeI |
17 | aptamer 12-1P | -- | -- |