Template:Kyoto/Notebook/Sep 8
From 2013.igem.org
(Difference between revisions)
(→Gel Extraction) |
(→Sep 8) |
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|experiment||9/8 Ptet-pT181 anti||1.6||9/8 spinach-DT XbaI+PstI||4.3|| | |experiment||9/8 Ptet-pT181 anti||1.6||9/8 spinach-DT XbaI+PstI||4.3|| | ||
|- | |- | ||
- | |experiment||9/6 Pcon SpeI+PstI||1.4||9/8 RBS- | + | |experiment||9/6 Pcon SpeI+PstI||1.4||9/8 RBS-laczα-DT XbaI+PstI||4.8|| |
|- | |- | ||
|experiment||9/8 Pcon-pT181 attenuator SpeI+PstI||3.4||9/7 aptamer12-1R-DT XbaI+PstI||1.9|| | |experiment||9/8 Pcon-pT181 attenuator SpeI+PstI||3.4||9/7 aptamer12-1R-DT XbaI+PstI||1.9|| | ||
Line 150: | Line 150: | ||
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 RBS-lysis1-DT XbaI+PstI||7.2|| | |experiment||9/8 Plac SpeI+PstI||4.3||9/7 RBS-lysis1-DT XbaI+PstI||7.2|| | ||
|- | |- | ||
- | |experiment||9/4 Plac SpeI+PstI||1.7||9/8 RBS- | + | |experiment||9/4 Plac SpeI+PstI||1.7||9/8 RBS-laczα-DT XbaI+PstI||4.8|| |
|} | |} | ||
</div> | </div> | ||
Line 242: | Line 242: | ||
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
- | ! ||8/20 J23100 RBS- | + | ! ||8/20 J23100 RBS-lacZα-DT-(1)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total |
|- | |- | ||
|2 cuts|||7µL||0.5µL||0.5µL||0µL||0µL||3µL||3µL||16µL||30µL | |2 cuts|||7µL||0.5µL||0.5µL||0µL||0µL||3µL||3µL||16µL||30µL | ||
Line 249: | Line 249: | ||
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
- | ! ||8/20 RBS- | + | ! ||8/20 RBS-lacZα-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total |
|- | |- | ||
|2 cuts||10µL||0.5µL||0.5µL||0µL||0µL||3µL||3µL||13µL||30µL | |2 cuts||10µL||0.5µL||0.5µL||0µL||0µL||3µL||3µL||13µL||30µL | ||
Line 265: | Line 265: | ||
|9/8 Pcont+aptamer 12-1R-DT||1||10||11||Amp | |9/8 Pcont+aptamer 12-1R-DT||1||10||11||Amp | ||
|- | |- | ||
- | |9/8 Pcon+RBS- | + | |9/8 Pcon+RBS-lacZα-DT||1||10||11||Amp |
|- | |- | ||
|9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT||1||10||11||Amp | |9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT||1||10||11||Amp | ||
Line 283: | Line 283: | ||
|9/8 Plux+RBS-lysis2-DT||1||10||11||CP | |9/8 Plux+RBS-lysis2-DT||1||10||11||CP | ||
|- | |- | ||
- | |9/8 Ptet+RBS- | + | |9/8 Ptet+RBS-lacZα-DT||1||10||11||CP |
|- | |- | ||
|9/8 Ptet-pT181 anti+spinach-DT||1||10||11||CP | |9/8 Ptet-pT181 anti+spinach-DT||1||10||11||CP | ||
Line 336: | Line 336: | ||
|1||spinach-DT|| || | |1||spinach-DT|| || | ||
|- | |- | ||
- | |2||J23100-RBS- | + | |2||J23100-RBS-lacZα-DT||EcoRI||SpeI |
|- | |- | ||
- | |3||J23100-RBS- | + | |3||J23100-RBS-lacZα-DT|| || |
|- | |- | ||
|4||100bp ladder|| || | |4||100bp ladder|| || | ||
|- | |- | ||
- | |5||RBS- | + | |5||RBS-lacZα-DT||EcoRI||SpeI |
|- | |- | ||
- | |6||RBS- | + | |6||RBS-lacZα-DT|| || |
|- | |- | ||
|7||RBS-lysis1-DT||XbaI||PstI | |7||RBS-lysis1-DT||XbaI||PstI | ||
Line 463: | Line 463: | ||
|1||100bp ladder|| | |1||100bp ladder|| | ||
|- | |- | ||
- | |2||J23100-RBS- | + | |2||J23100-RBS-laczα-DT||EcoRI+SpeI |
|- | |- | ||
- | |3||J23100-RBS- | + | |3||J23100-RBS-laczα-DT||EcoRI+SpeI |
|- | |- | ||
- | |5||RBS- | + | |5||RBS-laczα-DT||EcoRI+SpeI |
|- | |- | ||
- | |6||RBS- | + | |6||RBS-laczα-DT||EcoRI+SpeI |
|- | |- | ||
|8||RBS-lysis1-DT||XbaI+PstI | |8||RBS-lysis1-DT||XbaI+PstI | ||
Line 485: | Line 485: | ||
!Name||quantity||concentration[µg/mL]||260/280||260/230 | !Name||quantity||concentration[µg/mL]||260/280||260/230 | ||
|- | |- | ||
- | |Pcon-RBS- | + | |Pcon-RBS-laczα-DT(EcoRI+SpeI)|| ||5.6||2.03||0.25 |
|- | |- | ||
- | |RBS- | + | |RBS-laczα-DT(EcoRI+SpeI)|| ||4.4||2.04||0.33 |
|- | |- | ||
|RBS-lysis1-DT(XbaI+PstI)|| ||5.9||2.22||0.53 | |RBS-lysis1-DT(XbaI+PstI)|| ||5.9||2.22||0.53 | ||
Line 538: | Line 538: | ||
|experiment||Ptet-pT181 anti(SpeI+PstI)||30.6µg/mL||spin-DT(XbaI+PstI)||10.1µg/mL | |experiment||Ptet-pT181 anti(SpeI+PstI)||30.6µg/mL||spin-DT(XbaI+PstI)||10.1µg/mL | ||
|- | |- | ||
- | |experiment||Pcon-pT181 attenuator(SpeI+PstI)||14.5µg/mL||RBS- | + | |experiment||Pcon-pT181 attenuator(SpeI+PstI)||14.5µg/mL||RBS-laczα-DT(XbaI+PstI)||4.4µg/mL |
|- | |- | ||
|} | |} |
Revision as of 18:48, 25 September 2013
Contents |
Sep 8
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | RBS-lysis1-DT | XbaI & PstI |
3 | RBS-lysis1-DT | XbaI & PstI |
5 | RBS-lysis3-DT | XbaI & PstI |
6 | RBS-lysis3-DT | XbaI & PstI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
RBS-lysis1-DT (XbaI & PstI) | 5.3 | 1.98 | 0.06 |
RBS-lysis3-DT (XbaI & PstI) | 5.3 | 1.98 | 0.06 |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
3 | Pcon-pT181 attenuator | EcoRI & SpeI |
4 | Pcon-pT181 attenuator | EcoRI & SpeI |
6 | Spinach-DT | XbaI & PstI |
7 | Spinach-DT | XbaI & PstI |
9 | RBS-lacZα-DT | XbaI & PstI |
10 | RBS-lacZα-DT | XbaI & PstI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pcon-pT181 attenuator (EcoRI & SpeI) | 5.2 | 1.82 | 0.36 |
Spinach-DT (XbaI & PstI) | 8.0 | 1.87 | 0.25 |
RBS-lacZα-DT (XbaI & PstI) | 10.3 | 1.71 | 0.54 |
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | |
2 | Ptet-pT181 antisense | EcoRI & PstI |
3 | Ptet-pT181 antisense | EcoRI & PstI |
5 | pSB4K5 | EcoRI & SpeI |
6 | pSB4K5 | EcoRI & SpeI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Ptet-pT181 antisense(EcoRI & PstI) | 30.6 | 1.84 | 1.16 |
pSB4K5 (EcoRI & SpeI) | 21.8 | 1.87 | 0.98 |
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | |
2 | Pcon-pT181 attenuator | SpeI & PstI |
3 | Pcon-pT181 attenuator | SpeI & PstI |
5 | Plac | SpeI & PstI |
6 | Plac | SpeI & PstI |
8 | pSB1C3 | EcoRI & SpeI |
9 | pSB1C3 | EcoRI & SpeI |
11 | pSB1C3 | XbaI & PstI |
12 | pSB1C3 | XbaI & PstI |
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pcon-pT181 attenuator (SpeI & PstI) | 14.5 | 1.91 | 1.09 |
Plac (SpeI & PstI) | 11.6 | 1.74 | 1.19 |
pSB1C3 (EcoRI & SpeI) | 5.7 | 1.74 | 0.34 |
pSB1C3 (XbaI & PstI) | 5.6 | 2.00 | 0.37 |
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
Pcon-pT181 anti | 243.0 | 1.90 | 1.81 |
Pcon-Spinach-DT | 253.7 | 1.57 | 1.51 |
Ligation
state | Vector(µL) | Inserter(µL) | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/3 pT181 attenuator | 3.1 | |
experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/7 aptamer12-1R-DT XbaI+PstI | 2.1 | |
experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/8 spinach-DT XbaI+PstI | 4.6 | |
experiment | 9/6 Plac SpeI+PstI | 2 | 9/7 RBS-lysis2-DT XbaI+PstI | 10 | |
experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/7 RBS-lysis2-DT XbaI+PstI | 10 | |
experiment | 9/8 Ptet-pT181 anti | 1.6 | 9/8 spinach-DT XbaI+PstI | 4.3 | |
experiment | 9/6 Pcon SpeI+PstI | 1.4 | 9/8 RBS-laczα-DT XbaI+PstI | 4.8 | |
experiment | 9/8 Pcon-pT181 attenuator SpeI+PstI | 3.4 | 9/7 aptamer12-1R-DT XbaI+PstI | 1.9 | |
experiment | 9/6 Pcon SpeI+PstI | 1.4 | 9/7 aptamer12-1R-DT XbaI+PstI | 2.1 | |
experiment | 9/8 DT | 3.0 | 9/8 Pcon-pT181 attenuator EcoRI+SpeI | 7.9 | |
experiment | 9/6 Plux SpeI+PstI | 2 | 9/7 RBS-lysis1-DT XbaI+PstI | 7.2 | |
experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/7 RBS-lysis1-DT XbaI+PstI | 7.2 | |
experiment | 9/4 Plac SpeI+PstI | 1.7 | 9/8 RBS-laczα-DT XbaI+PstI | 4.8 |
Restriction Enzyme Digestion
9/8 Pcon-pT181 anti | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 8.2µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 13.8µL | 30µL |
NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |
9/8 Pcon-spin-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 7.9µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 14.1µL | 30µL |
NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |
8/21 F1 atte | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 6.5µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 15.5µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
8/21 F3m2 atte | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 6.0µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 16µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
8/21 F1 anti | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 6.7µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 15.3µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
8/21 F6 anti | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 6.0µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 16µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
8/21 tetR aptamer 12_1M | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 5.3µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 16.7µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
8/21 tetR aptamer 12_P | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 13.3µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 8.7µL | 30µL |
NC | 0.7µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |
9/4 spin-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 5.4µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 16.6µL | 30µL |
NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |
9/7 RBS-lysis1-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 10µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
9/6 RBS-lysis2-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 13µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 9µL | 30µL |
NC | 0.7µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |
9/7 RBS-lysis3-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 10µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
8/20 J23100 RBS-lacZα-DT-(1) | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 7µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 16µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
8/20 RBS-lacZα-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2 cuts | 10µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 13µL | 30µL |
NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
Transformation
Name | Sample(µL) | Competent Cells(µL) | Total(µL) | Plate |
---|---|---|---|---|
9/8 Pcont+aptamer 12-1R-DT | 1 | 10 | 11 | Amp |
9/8 Pcon+RBS-lacZα-DT | 1 | 10 | 11 | Amp |
9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT | 1 | 10 | 11 | Amp |
9/8 Plac+RBS-lysis1-DT | 2 | 20 | 22 | CP |
9/8 Plac+RBS-lysis2-DT | 2 | 20 | 22 | CP |
9/8 Plac+aptamer 12-1R-DT | 2 | 20 | 22 | CP |
9/8 Plac+spinach | 2 | 20 | 22 | CP |
9/8 Plac+pT181 attenuator | 2 | 20 | 22 | CP |
9/8 Plux+RBS-lysis1-DT | 1 | 10 | 11 | CP |
9/8 Plux+RBS-lysis2-DT | 1 | 10 | 11 | CP |
9/8 Ptet+RBS-lacZα-DT | 1 | 10 | 11 | CP |
9/8 Ptet-pT181 anti+spinach-DT | 1 | 10 | 11 | CP |
9/8 Pcon-pT181 attenuator+DT | 1 | 10 | 11 | CP |
Electrophoresis
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 100bp ladder | ||
2 | Pcon-spin-DT | EcoRI | SpeI |
3 | Pcon-spin-DT | ||
4 | Fusion1 attenuator | XbaI | PstI |
5 | Fusion1 attenuator | ||
6 | Fusion3m2 attenuator | XbaI | PstI |
7 | Fusion3m2 attenuator | ||
8 | Fusion1 anti | XbaI | PstI |
9 | Fusion1 anti | ||
10 | Fusion6 anti | XbaI | PstI |
11 | Fusion6 anti | ||
12 | aptamer 12-1M | XbaI | PstI |
13 | aptamer 12-1M | ||
14 | aptamer 12-P | XbaI | PstI |
15 | aptamer 12-P | ||
16 | spinach-DT | XbaI | PstI |
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | spinach-DT | ||
2 | J23100-RBS-lacZα-DT | EcoRI | SpeI |
3 | J23100-RBS-lacZα-DT | ||
4 | 100bp ladder | ||
5 | RBS-lacZα-DT | EcoRI | SpeI |
6 | RBS-lacZα-DT | ||
7 | RBS-lysis1-DT | XbaI | PstI |
8 | RBS-lysis1-DT |
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 100bp ladder | ||
2 | RBS-lysis2-DT | XbaI | PstI |
3 | RBS-lysis2-DT | ||
4 | RBS-lysis3-DT | XbaI | PstI |
5 | RBS-lysis3-DT | ||
6 | Pcon pT181 anti | SpeI | PstI |
7 | Pcon pT181 anti |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | Pcon-spin-DT | EcoRI+SpeI |
3 | Pcon-Spin-DT | EcoRI+SpeI |
5 | Fusion 1 attenuator | XbaI+PstI |
6 | Fusion 1 attenuator | XbaI+PstI |
8 | Fusion3m2 attenuator | XbaI+PstI |
9 | Fusion3m2 attenuator | XbaI+PstI |
11 | Fusion 1 anti | XbaI+PstI |
12 | Fusion 1 anti | XbaI+PstI |
Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|---|
Pcon-spin-DT(EcoRI+SpeI) | 10.5 | 1.86 | 0.39 | |
Fusion 1 attenuator(XbaI+PstI) | 7.0 | 1.71 | 0.27 | |
Fusion3m2 attenuator(XbaI+PstI) | 8.6 | 1.75 | 0.47 | |
Fusion 1 anti(XbaI+PstI) | 7.6 | 1.87 | 0.43 |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | Fusion6 anti | XbaI+PstI |
3 | Fusion6 anti | XbaI+PstI |
5 | aptamer12_1M | XbaI+PstI |
6 | aptamer12_1M | XbaI+PstI |
Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|---|
Fusion6 anti(XbaI+PstI) | 5.0 | 1.76 | 0.20 | |
aptamer12_1M(XbaI+PstI) | 3.3 | 2.31 | 0.04 |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | aptamer12_P | XbaI+PstI |
3 | aptamer12_P | XbaI+PstI |
5 | spinach-DT | EcoRI+SpeI |
6 | spinach-DT | EcoRI+SpeI |
Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|---|
aptamer12_P(XbaI+PstI) | 3.4 | 1.72 | 0.26 | |
spinach-DT(EcoRI+SpeI) | 10.1 | 1.57 | 0.47 |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | J23100-RBS-laczα-DT | EcoRI+SpeI |
3 | J23100-RBS-laczα-DT | EcoRI+SpeI |
5 | RBS-laczα-DT | EcoRI+SpeI |
6 | RBS-laczα-DT | EcoRI+SpeI |
8 | RBS-lysis1-DT | XbaI+PstI |
9 | RBS-lysis1-DT | XbaI+PstI |
11 | RBS-lysis2-DT | XbaI+PstI |
12 | RBS-lysis2-DT | XbaI+PstI |
Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|---|
Pcon-RBS-laczα-DT(EcoRI+SpeI) | 5.6 | 2.03 | 0.25 | |
RBS-laczα-DT(EcoRI+SpeI) | 4.4 | 2.04 | 0.33 | |
RBS-lysis1-DT(XbaI+PstI) | 5.9 | 2.22 | 0.53 | |
RBS-lysis2-DT(XbaI+PstI) | 5.1 | 1.67 | 0.48 |
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | |
2 | RBS-lysis3-DT | XbaI+PstI |
3 | RBS-lysis3-DT | XbaI+PstI |
5 | Pcon-pT181 anti | SpeI+PstI |
6 | Pcon-pT181 anti | SpeI+PstI |
Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|---|
RBS-lysis3-DT(XbaI+PstI) | 23.4 | 1.72 | 0.88 | |
Pcon-pT181 anti(SpeI+PstI) | 31.3 | 1.86 | 0.92 |
Ligation
state | Vector | Inserter | ||
---|---|---|---|---|
experiment | Pcon-spin-DT(EcoRI&SpeI) | 10.5µg/mL | pSB6A1(EcoRI&SpeI) | 8.9µg/mL |
experiment | Pcon-spin-DT(EcoRI&SpeI) | 10.5µg/mL | pSB4K5(EcoRI&SpeI) | 21.8µg/mL |
experiment | Pcon-pT181 anti(SpeI+PstI) | 31.3µg/mL | spin-DT(XbaI+PstI) | 10.1µg/mL |
experiment | Plac(SpeI+PstI) | 11.6µg/mL | RBS-lysis3-DT | 23.4µg/mL |
experiment | Plux(SpeI+PstI) | 25µg/mL | RBS-lysis3-DT | 23.4µg/mL |
experiment | Ptet-pT181 anti(SpeI+PstI) | 30.6µg/mL | spin-DT(XbaI+PstI) | 10.1µg/mL |
experiment | Pcon-pT181 attenuator(SpeI+PstI) | 14.5µg/mL | RBS-laczα-DT(XbaI+PstI) | 4.4µg/mL |
PCR
KaiA(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(661-RBS-KaiA-fwd) | Primer(kaiA-662-rev-ver2) | MilliQ | total |
---|---|---|---|---|---|---|---|---|
1 | 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 | 10s | 30s | 28s | 30cycles |
KaiB(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(662-RBS-KaiB-fwd-ver2) | Primer(kaiB-663-rev-ver2) | MilliQ | total |
---|---|---|---|---|---|---|---|---|
1 | 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 | 10s | 30s | 28s | 30cycles |
DT(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(664-DT-fwd-ver2) | Primer(DT-suffix-660-rev-ver2) | MilliQ | total |
---|---|---|---|---|---|---|---|---|
0.1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.4 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2min | 10s | 30s | 28s | 30cycles |
P-KaiB(PCR prpduct) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(prefix-PkaiBC-fwd-ver2) | Primer(PkaiBC-suffix--662-rev-ver2) | MilliQ | total |
---|---|---|---|---|---|---|---|---|
0.7 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.8 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2min | 10s | 30s | 28s | 30cycles |
Plac(1)(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(GG0-pSBIC3-fwd-ver2) | Primer(Plac-GG1-rev-ver2) | MilliQ | total |
---|---|---|---|---|---|---|---|---|
0.13 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.37 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2min | 10s | 30s | 60s | 30cycles |
KaiC(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(GG3-RBS-KaiC-fwd-ver2) | Primer(KaiC-GG4-rev-ver2) | MilliQ | total |
---|---|---|---|---|---|---|---|---|
1 | 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 | 10s | 30s | 60s | 30cycles |
Gel Extraction
Lane | DNA |
---|---|
1 | 100bp ladder |
2 | KaiA |
3 | KaiA |
4 | KaiB |
5 | KaiB |
6 | DT |
7 | DT |
8 | P-KaiBC |
9 | P-KaiBC |
10 | nega |
11 | 100bp ladder |
File:Xxxbeforexxx.xxx
File:Xxxafterxxx.xxx
Lane | DNA |
---|---|
1 | 100bp ladder |
2 | KaiA |
3 | KaiA |
4 | KaiB |
5 | KaiB |
6 | DT |
7 | DT |
8 | P-KaiBC |
9 | P-KaiBC |
10 | nega |
11 | 100bp ladder |
File:Xxxbeforxxx.xxx
File:Xxxafterxxx.xxx
Lane | DNA |
---|---|
1 | Plac |
2 | Plac |
3 | KaiC |
4 | KaiC |
5 | nega |
6 | 1kb ladder |