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Template:Kyoto/Notebook/Sep 14
From 2013.igem.org
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===Restriction Enzyme Digestion=== | ===Restriction Enzyme Digestion=== | ||
<div class="experiment"> | <div class="experiment"> | ||
| - | <span class="author"> | + | <span class="author">Hirano</span> |
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | ! ||9/14 pSB4K5S||EcoRI||XbaI||SpeI||PstI||BufferB||BufferD||BSA||MilliQ||total |
|- | |- | ||
| - | | | + | |2cuts||10µL||1µL||0µL||1µL||0µL||3µL||0µL||3µL||12µL||30µL |
|- | |- | ||
| - | |NC | + | |NC||0.5µL||0µL||0µL||0µL||0µL||1µL||0µL||1µL||7.5µL||10µL |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | ! ||9/13 Plac||XbaI||PstI||BufferH||BSA||MilliQ||total |
|- | |- | ||
| - | | | + | |1 cut||9µL||0µL||1µL||3µL||3µL||14µL||30µL |
|- | |- | ||
| - | |NC|| | + | |NC||1µL||0µL||0µL||1µL||1µL||7µL||10µL |
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | ! ||9/14 PKaiBC||EcoRI||XbaI||SpeI||PstI||BufferB||BSA||MilliQ||total |
|- | |- | ||
| - | | | + | |2cuts||11µL||1µL||0µL||1µL||0µL||3µL||3µL||11µL||30µL |
|- | |- | ||
| - | |NC|| | + | |NC||4µL||0µL||0µL||0µL||0µL||1µL||1µL||4µL||10µL |
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | ! ||9/14 RpaB||EcoRI||XbaI||SpeI||PstI||BufferD||BSA||MilliQ||total |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
|- | |- | ||
| - | | | + | |2cuts||13µL||0µL||1µL||0µL||1µL||3µL||3µL||9µL||30µL |
|- | |- | ||
| - | |NC|| | + | |NC||2µL||0µL||0µL||0µL||0µL||1µL||1µL||5µL||10µL |
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! ||8/ | + | ! ||8/17 RBS-GFP-DT||EcoRI||XbaI||SpeI||PstI||BufferD||BSA||MilliQ||total |
|- | |- | ||
| - | | | + | |1cut||9µL||0µL||1µL||0µL||0µL||3µL||3µL||14µL||30µL |
|- | |- | ||
| - | |NC | + | |NC||0.5µL||0µL||0µL||0µL||0µL||1µL||1µL||7.5µL||10µL |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
|} | |} | ||
*incubated 37°C 1hour | *incubated 37°C 1hour | ||
| - | |||
</div> | </div> | ||
===Liquid Culture=== | ===Liquid Culture=== | ||
| - | |||
<div class="experiment"> | <div class="experiment"> | ||
| - | <span class="author"> | + | <span class="author">Hirano</span> |
{| class="wikitable" | {| class="wikitable" | ||
!Sample||medium | !Sample||medium | ||
|- | |- | ||
| - | | | + | |9/11 Plac(BBa-R0011)-(3)||Plusgrow medium(+Amp) |
| + | |} | ||
| + | </div> | ||
| + | |||
| + | ===Electrophoresis=== | ||
| + | |||
| + | <div class="experiment"> | ||
| + | <span class="author">Hirano</span> | ||
| + | {| class="wikitable" | ||
| + | !Lane||Sample||Enzyme1||Enzyme2 | ||
|- | |- | ||
| - | | | + | |1||1kb ladder||--||-- |
|- | |- | ||
| - | | | + | |2||pSB4K5(1)||EcoRI||SpeI |
|- | |- | ||
| - | | | + | |3|| pSB4K5(1)||--||-- |
|- | |- | ||
| - | | | + | |4||Plac(1A2)||PstI||-- |
|- | |- | ||
| - | | | + | |5||Plac(1A2)||--||-- |
|- | |- | ||
| - | | | + | |6||PKaiBC||EcoRI||SpeI |
|- | |- | ||
| - | | | + | |7||PKaiBC||--||-- |
|- | |- | ||
| - | | | + | |8||RpaB(1)||XbaI||PstI |
| + | |- | ||
| + | |9||RpaB(1)||--||-- | ||
| + | |- | ||
| + | |10||RBS-GFP-DT||XbaI||-- | ||
| + | |- | ||
| + | |11||RBS-GFP-DT||--||-- | ||
| + | |- | ||
| + | |12||1kb ladder||--||-- | ||
|} | |} | ||
| + | [[File:igku_0914_E1.jpg]]<br> | ||
</div> | </div> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! | + | !Lane||Sample||Enzyme |
|- | |- | ||
| - | + | |1||1kb ladder||-- | |
| - | + | ||
| - | + | ||
| - | + | ||
|- | |- | ||
| - | | | + | |2||Plac(pSB1A2)||PstI |
|- | |- | ||
| - | | | + | |3||Plac(pSB1A2)||-- |
|} | |} | ||
| - | < | + | [[File:igku_0914_E2.jpg]]<br> |
| - | + | ||
| - | === | + | ===Sequence PCR=== |
<div class="experiment"> | <div class="experiment"> | ||
<span class="author">No name</span> | <span class="author">No name</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | !genome DNA||Big Dye||5x buffer||temp(400mg)||primer||milliQ |
|- | |- | ||
| - | | | + | |Pcon-RBS-luxR-DT||0.5||1.75||0.9||0.5||6.85 |
|- | |- | ||
| - | | | + | |Pcon-RBS-luxR-DT||0.5||1.75||0.4||0.5||7.35 |
|- | |- | ||
| - | | | + | |Pcon-RBS-tetR-DT||0.5||1.75||3||0.5||5.25 |
|- | |- | ||
| - | | | + | |Pcon-RBS-tetR-DT||0.5||1.75||3||0.5||5.25 |
| - | + | ||
| - | + | ||
| - | + | ||
|- | |- | ||
| - | | | + | |Plux||0.5||1.75||2.4||0.5||5.35 |
|- | |- | ||
| - | | | + | |Plux-RBS-GFP-DT||0.5||1.75||1.9||0.5||5.85 |
| + | |- | ||
| + | |Plux-RBS-GFP-DT||0.5||0.5||1.9||0.5||5.85 | ||
| + | |- | ||
| + | |Pcon-RBS-GFP-DT||0.5||0.5||1.2||0.5||6.55 | ||
| + | |- | ||
| + | |Pcon-RBS-GFP-DT||0.5||0.5||1.2||0.5||6.55 | ||
| + | |- | ||
| + | |RBS-lysis3-DT||0.5||0.5||1.0||0.5||6.75 | ||
| + | |- | ||
| + | |Pcon-antisense-Spinach-DT||0.5||0.5||1.4||0.5||6.35 | ||
| + | |- | ||
| + | |Pcon-RBS lacZα-DT||0.5||0.5||1.4||0.5||6.35 | ||
| + | |- | ||
| + | |Ptet-RBS lacZα-DT||0.5||0.5||0.59||0.5||1.85 | ||
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | !PreDenature||Denature||Annealing||Extension||cycle |
|- | |- | ||
| - | | | + | |96°C||96°C||50°C||60°C||-- |
|- | |- | ||
| - | | | + | |2min||10sec||5sec||4min||30cycle |
|} | |} | ||
| + | </div> | ||
| + | |||
| + | ===Gel Extraction=== | ||
| + | <div class="experiment"> | ||
| + | <span class="author">Tatsui</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | !Lane||DNA||Enzyme |
|- | |- | ||
| - | | | + | |1||1kb ladder||-- |
|- | |- | ||
| - | | | + | |2||pSB4K5||EcoRI&SpeI |
|- | |- | ||
| - | + | |3||pSB4K5||EcoRI&SpeI | |
|- | |- | ||
| - | | | + | |4||pSB4K5||EcoRI&SpeI |
|} | |} | ||
| + | [[File:igku_0914_G8.jpg]]<br> | ||
| + | [[File:igku_0914_G9.jpg]]<br> | ||
| + | |||
| + | ===Restriction Enzyme Digestion=== | ||
| + | <div class="experiment"> | ||
| + | <span class="author">No name</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! || | + | ! ||9/14 Plac(1A2)||EcoRI||XbaI||SpeI||PstI||BufferB||BSA||MilliQ||total |
|- | |- | ||
| - | | | + | |2cuts||23µL||0µL||0µL||0.3µL||0µL||3µL||3µL||0µL||30µL |
| + | |} | ||
| + | {| class="wikitable" | ||
| + | ! ||9/14 RBS-GFP-DT||EcoRI||XbaI||SpeI||PstI||BufferH||BSA||MilliQ||total | ||
|- | |- | ||
| - | + | |2 cuts||23µL||1µL||0µL||0µL||0µL||3µL||3µL||0µL||30µL | |
| - | + | ||
| - | |2 cuts|| | + | |
| - | + | ||
| - | + | ||
|} | |} | ||
| - | |||
| - | |||
</div> | </div> | ||
| - | |||
===Ligation=== | ===Ligation=== | ||
| Line 161: | Line 174: | ||
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2 | !state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2 | ||
|- | |- | ||
| - | |experiment|| | + | |experiment||9/13 Pcon-PT181attenuator(SpeI&PstI)||1.7||9/13 RBS-lacZα-DT (XbaI & PstI)||12.5||5 |
|- | |- | ||
| - | |experiment|| | + | |experiment||9/13 pSB2C3 (XbaI & PstI)||1.9||9/14 RpaB (XbaI & PstI)||16.3||10.5 |
|- | |- | ||
| - | |experiment|| | + | |experiment||9/13 pSB2C3 (XbaI & PstI)||4.7||9/14 PkaiBC (EcoRI & SpeI)||6.7||5.7 |
|- | |- | ||
| - | |experiment|| | + | |experiment||9/14 RBS-GFP-DT (PstI&XbaI)||0.7||9/14 PkaiBC (EcoRI & SpeI)||2.6||1.6 |
|- | |- | ||
| - | |experiment|| | + | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis1-DT (XbaI & PstI)||4.9||3.6 |
|- | |- | ||
| - | |experiment|| | + | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis2-DT (XbaI & PstI)||7.0||4.7 |
|- | |- | ||
| - | |experiment|| | + | |experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis3-DT (XbaI & PstI)||9.2||5.8 |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
|} | |} | ||
Samples were evaporeted used evaporator into about 7 µL | Samples were evaporeted used evaporator into about 7 µL | ||
</div> | </div> | ||
| - | |||
===Colony PCR=== | ===Colony PCR=== | ||
| Line 192: | Line 198: | ||
!Sample||base pair | !Sample||base pair | ||
|- | |- | ||
| - | | | + | |9/13 Plux＋RBS-lysis1-DT (1)~(3)||-- |
| + | |- | ||
| + | |9/13 Plux＋RBS-lysis2-DT (1)~(3)||- | ||
| + | |- | ||
| + | |9/13 Plux＋RBS-lysis3-DT (1)~(2)||- | ||
| + | |} | ||
| + | {| class="wikitable" | ||
| + | !Sample||base pair | ||
|- | |- | ||
| - | | | + | |9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)||-- |
|- | |- | ||
| - | | | + | |9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)||- |
|- | |- | ||
| - | | | + | |9/13 Plac＋PT181attenuator (1)||- |
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! | + | !Sample||base pair |
|- | |- | ||
| - | | | + | |Dλ-lux(1) (1)~(2)||-- |
|- | |- | ||
| - | | | + | |NC ||-- |
|} | |} | ||
</div> | </div> | ||
| - | === | + | ===Electrophoresis=== |
| - | + | ||
<div class="experiment"> | <div class="experiment"> | ||
<span class="author">No name</span> | <span class="author">No name</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | !Sample|| | + | !Lane||Sample |
| + | |- | ||
| + | |1||1kbp ladder | ||
| + | |- | ||
| + | |2||9/13 Plux+RBS-lysis1-DT1 | ||
| + | |- | ||
| + | |3||9/13 Plux+RBS-lysis1-DT2 | ||
| + | |- | ||
| + | |4||9/13 Plux+RBS-lysis1-DT3 | ||
| + | |- | ||
| + | |5||9/13 Plux+RBS-lysis2-DT1 | ||
| + | |- | ||
| + | |6||9/13 Plux+RBS-lysis2-DT2 | ||
| + | |- | ||
| + | |7||9/13 Plux+RBS-lysis2-DT3 | ||
| + | |- | ||
| + | |8||9/13 Plux+RBS-lysis3-DT1 | ||
|- | |- | ||
| - | | | + | |9||9/13 Plux+RBS-lysis3-DT2 |
|- | |- | ||
| - | | | + | |10||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1 |
|- | |- | ||
| - | | | + | |11||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2 |
|- | |- | ||
| - | | | + | |12||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3 |
|- | |- | ||
| - | | | + | |13||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4 |
|- | |- | ||
| - | | | + | |14||9/13 Ptet-pT181 antisense+Spinach-DT1 |
|- | |- | ||
| - | | | + | |15||9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1 |
|- | |- | ||
| - | | | + | |16||9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1 |
|- | |- | ||
| - | | | + | |17||9/13 Plac+pT181 attenuator |
| + | |} | ||
| + | [[File:Igku_0914_E5.jpg]]<br> | ||
| + | </div> | ||
| + | |||
| + | {| class="wikitable" | ||
| + | !Lane||Sample | ||
| + | |- | ||
| + | |1||1kbp ladder | ||
| + | |- | ||
| + | |2||Pλ-lux(1)1 | ||
| + | |- | ||
| + | |3|| Pλ-lux(1)1 | ||
| + | |- | ||
| + | |4||NC | ||
| + | |} | ||
| + | [[File:Igku_0914_E6.jpg]]<br> | ||
| + | |||
| + | ===Liquid Culture=== | ||
| + | <div class="experiment"> | ||
| + | <span class="author">No name</span> | ||
| + | {| class="wikitable" | ||
| + | !Sample||medium | ||
| + | |- | ||
| + | |9/13 Pλ-luxI(1)-1||Plusgrow medium(+Amp) | ||
|} | |} | ||
</div> | </div> | ||
| Line 238: | Line 291: | ||
===Transformation=== | ===Transformation=== | ||
<div class="experiment"> | <div class="experiment"> | ||
| - | <span class="author"> | + | <span class="author">Nakamoto</span> |
{| class="wikitable" | {| class="wikitable" | ||
!Name||Sample||Competent Cells||Total||Plate | !Name||Sample||Competent Cells||Total||Plate | ||
|- | |- | ||
| - | | | + | |9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)||2µL||20µL||22µL||Amp |
|- | |- | ||
| - | | | + | |9/13 SB2C3＋9/14 PzαB||2µL||20µL||22µL||CP |
|- | |- | ||
| - | | | + | |9/13 pSB2C3＋9/14 PkaiBC||2µL||20µL||22µL||CP |
|- | |- | ||
| - | | | + | |9/13 RBS-GFP-DT＋9/14 PkaiBC||2µL||20µL||22µL||CP |
|- | |- | ||
| - | | | + | |Plac＋RBS-lysis1-DT||2µL||20µL||22µL||Amp |
|- | |- | ||
| - | | | + | |Plac＋RBS-lysis2-DT||2µL||20µL||22µL||Amp |
|- | |- | ||
| - | | | + | |Plac＋RBS-lysis2-DT||2µL||20µL||22µL||Amp |
|} | |} | ||
| - | |||
| - | |||
| - | |||
| - | |||
</div> | </div> | ||
| - | + | ===Sequence PCR=== | |
| - | ===PCR=== | + | |
<div class="experiment"> | <div class="experiment"> | ||
| - | <span class="author"> | + | <span class="author">Tatsui</span> |
{| class="wikitable" | {| class="wikitable" | ||
| - | !genome DNA|| | + | !genome DNA||temp||Big Dye||5x buffer||primer||MilliQ |
| + | |- | ||
| + | |9/6 PT181 attenuator(VR)||3.9||0.5||1.75||0.5||3.85 | ||
| + | |- | ||
| + | |8/17 RBS lacZα-DT||2.1||0.5||1.75||0.5||5.65 | ||
|- | |- | ||
| - | | || | + | |9/6 PT181 attenuator(VR)||3.9||0.5||1.75||0.5||3.85 |
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
!PreDenature||Denature||Annealing||Extension||cycle | !PreDenature||Denature||Annealing||Extension||cycle | ||
|- | |- | ||
| - | | | + | |96°C||50°C||60°C||4°C||-- |
|- | |- | ||
| - | | | + | |10sec||5sec||4min||1min||30cycle |
|} | |} | ||
</div> | </div> | ||
Latest revision as of 17:41, 27 September 2013
Contents |
Sep 14
Restriction Enzyme Digestion
| 9/14 pSB4K5S | EcoRI | XbaI | SpeI | PstI | BufferB | BufferD | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|---|
| 2cuts | 10µL | 1µL | 0µL | 1µL | 0µL | 3µL | 0µL | 3µL | 12µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 1µL | 7.5µL | 10µL |
| 9/13 Plac | XbaI | PstI | BufferH | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 1 cut | 9µL | 0µL | 1µL | 3µL | 3µL | 14µL | 30µL |
| NC | 1µL | 0µL | 0µL | 1µL | 1µL | 7µL | 10µL |
| 9/14 PKaiBC | EcoRI | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cuts | 11µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 11µL | 30µL |
| NC | 4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 4µL | 10µL |
| 9/14 RpaB | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cuts | 13µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 9µL | 30µL |
| NC | 2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5µL | 10µL |
| 8/17 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 1cut | 9µL | 0µL | 1µL | 0µL | 0µL | 3µL | 3µL | 14µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
- incubated 37°C 1hour
Liquid Culture
| Sample | medium |
|---|---|
| 9/11 Plac(BBa-R0011)-(3) | Plusgrow medium(+Amp) |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 1kb ladder | -- | -- |
| 2 | pSB4K5(1) | EcoRI | SpeI |
| 3 | pSB4K5(1) | -- | -- |
| 4 | Plac(1A2) | PstI | -- |
| 5 | Plac(1A2) | -- | -- |
| 6 | PKaiBC | EcoRI | SpeI |
| 7 | PKaiBC | -- | -- |
| 8 | RpaB(1) | XbaI | PstI |
| 9 | RpaB(1) | -- | -- |
| 10 | RBS-GFP-DT | XbaI | -- |
| 11 | RBS-GFP-DT | -- | -- |
| 12 | 1kb ladder | -- | -- |
| Lane | Sample | Enzyme |
|---|---|---|
| 1 | 1kb ladder | -- |
| 2 | Plac(pSB1A2) | PstI |
| 3 | Plac(pSB1A2) | -- |
Sequence PCR
| genome DNA | Big Dye | 5x buffer | temp(400mg) | primer | milliQ |
|---|---|---|---|---|---|
| Pcon-RBS-luxR-DT | 0.5 | 1.75 | 0.9 | 0.5 | 6.85 |
| Pcon-RBS-luxR-DT | 0.5 | 1.75 | 0.4 | 0.5 | 7.35 |
| Pcon-RBS-tetR-DT | 0.5 | 1.75 | 3 | 0.5 | 5.25 |
| Pcon-RBS-tetR-DT | 0.5 | 1.75 | 3 | 0.5 | 5.25 |
| Plux | 0.5 | 1.75 | 2.4 | 0.5 | 5.35 |
| Plux-RBS-GFP-DT | 0.5 | 1.75 | 1.9 | 0.5 | 5.85 |
| Plux-RBS-GFP-DT | 0.5 | 0.5 | 1.9 | 0.5 | 5.85 |
| Pcon-RBS-GFP-DT | 0.5 | 0.5 | 1.2 | 0.5 | 6.55 |
| Pcon-RBS-GFP-DT | 0.5 | 0.5 | 1.2 | 0.5 | 6.55 |
| RBS-lysis3-DT | 0.5 | 0.5 | 1.0 | 0.5 | 6.75 |
| Pcon-antisense-Spinach-DT | 0.5 | 0.5 | 1.4 | 0.5 | 6.35 |
| Pcon-RBS lacZα-DT | 0.5 | 0.5 | 1.4 | 0.5 | 6.35 |
| Ptet-RBS lacZα-DT | 0.5 | 0.5 | 0.59 | 0.5 | 1.85 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96°C | 96°C | 50°C | 60°C | -- |
| 2min | 10sec | 5sec | 4min | 30cycle |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 1kb ladder | -- |
| 2 | pSB4K5 | EcoRI&SpeI |
| 3 | pSB4K5 | EcoRI&SpeI |
| 4 | pSB4K5 | EcoRI&SpeI |
Restriction Enzyme Digestion
| 9/14 Plac(1A2) | EcoRI | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2cuts | 23µL | 0µL | 0µL | 0.3µL | 0µL | 3µL | 3µL | 0µL | 30µL |
| 9/14 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferH | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|---|---|
| 2 cuts | 23µL | 1µL | 0µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | 9/13 Pcon-PT181attenuator(SpeI&PstI) | 1.7 | 9/13 RBS-lacZα-DT (XbaI & PstI) | 12.5 | 5 |
| experiment | 9/13 pSB2C3 (XbaI & PstI) | 1.9 | 9/14 RpaB (XbaI & PstI) | 16.3 | 10.5 |
| experiment | 9/13 pSB2C3 (XbaI & PstI) | 4.7 | 9/14 PkaiBC (EcoRI & SpeI) | 6.7 | 5.7 |
| experiment | 9/14 RBS-GFP-DT (PstI&XbaI) | 0.7 | 9/14 PkaiBC (EcoRI & SpeI) | 2.6 | 1.6 |
| experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis1-DT (XbaI & PstI) | 4.9 | 3.6 |
| experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis2-DT (XbaI & PstI) | 7.0 | 4.7 |
| experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis3-DT (XbaI & PstI) | 9.2 | 5.8 |
Samples were evaporeted used evaporator into about 7 µL
Colony PCR
| Sample | base pair |
|---|---|
| 9/13 Plux＋RBS-lysis1-DT (1)~(3) | -- |
| 9/13 Plux＋RBS-lysis2-DT (1)~(3) | - |
| 9/13 Plux＋RBS-lysis3-DT (1)~(2) | - |
| Sample | base pair |
|---|---|
| 9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4) | -- |
| 9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2) | - |
| 9/13 Plac＋PT181attenuator (1) | - |
| Sample | base pair |
|---|---|
| Dλ-lux(1) (1)~(2) | -- |
| NC | -- |
Electrophoresis
| Lane | Sample |
|---|---|
| 1 | 1kbp ladder |
| 2 | 9/13 Plux+RBS-lysis1-DT1 |
| 3 | 9/13 Plux+RBS-lysis1-DT2 |
| 4 | 9/13 Plux+RBS-lysis1-DT3 |
| 5 | 9/13 Plux+RBS-lysis2-DT1 |
| 6 | 9/13 Plux+RBS-lysis2-DT2 |
| 7 | 9/13 Plux+RBS-lysis2-DT3 |
| 8 | 9/13 Plux+RBS-lysis3-DT1 |
| 9 | 9/13 Plux+RBS-lysis3-DT2 |
| 10 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1 |
| 11 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2 |
| 12 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3 |
| 13 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4 |
| 14 | 9/13 Ptet-pT181 antisense+Spinach-DT1 |
| 15 | 9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1 |
| 16 | 9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1 |
| 17 | 9/13 Plac+pT181 attenuator |
| Lane | Sample |
|---|---|
| 1 | 1kbp ladder |
| 2 | Pλ-lux(1)1 |
| 3 | Pλ-lux(1)1 |
| 4 | NC |
Liquid Culture
| Sample | medium |
|---|---|
| 9/13 Pλ-luxI(1)-1 | Plusgrow medium(+Amp) |
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| 9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI) | 2µL | 20µL | 22µL | Amp |
| 9/13 SB2C3＋9/14 PzαB | 2µL | 20µL | 22µL | CP |
| 9/13 pSB2C3＋9/14 PkaiBC | 2µL | 20µL | 22µL | CP |
| 9/13 RBS-GFP-DT＋9/14 PkaiBC | 2µL | 20µL | 22µL | CP |
| Plac＋RBS-lysis1-DT | 2µL | 20µL | 22µL | Amp |
| Plac＋RBS-lysis2-DT | 2µL | 20µL | 22µL | Amp |
| Plac＋RBS-lysis2-DT | 2µL | 20µL | 22µL | Amp |
Sequence PCR
| genome DNA | temp | Big Dye | 5x buffer | primer | MilliQ |
|---|---|---|---|---|---|
| 9/6 PT181 attenuator(VR) | 3.9 | 0.5 | 1.75 | 0.5 | 3.85 |
| 8/17 RBS lacZα-DT | 2.1 | 0.5 | 1.75 | 0.5 | 5.65 |
| 9/6 PT181 attenuator(VR) | 3.9 | 0.5 | 1.75 | 0.5 | 3.85 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 96°C | 50°C | 60°C | 4°C | -- |
| 10sec | 5sec | 4min | 1min | 30cycle |
