Template:Kyoto/Notebook/Sep 13
From 2013.igem.org
(Difference between revisions)
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(→PCR) |
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|9/12 Plux-4||125.3||1.93||1.44 | |9/12 Plux-4||125.3||1.93||1.44 | ||
|- | |- | ||
- | |9/12 | + | |9/12 Fusion3n2 attenuator-1||231.9||1.81||1.31 |
|} | |} | ||
</div> | </div> | ||
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{| class="wikitable" | {| class="wikitable" | ||
Line 127: | Line 90: | ||
|4||pSB1C3||XbaI&PstI | |4||pSB1C3||XbaI&PstI | ||
|} | |} | ||
- | + | [[File:igku_9139.jpg]]<br> | |
- | [[File: | + | [[File:igku_91310.jpg]]<br> |
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===Restriction Enzyme Digestion=== | ===Restriction Enzyme Digestion=== | ||
Line 157: | Line 110: | ||
|1cut(PstI)||10||0µL||0µL||0µL||1µL||3µL||3µL||13µL||30µL | |1cut(PstI)||10||0µL||0µL||0µL||1µL||3µL||3µL||13µL||30µL | ||
|- | |- | ||
- | |NC||0.5||0µL | + | |NC||0.5||0µL||0µL||0µL||0µL||1µL||1µL||7.5µL||10µL |
|} | |} | ||
Line 244: | Line 197: | ||
|} | |} | ||
- | [[File: | + | [[File:igku_91311.jpg]]<br> |
- | [[File: | + | [[File:igku_91312.jpg]]<br> |
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===Liquid Culture=== | ===Liquid Culture=== | ||
Line 291: | Line 235: | ||
|1cut(SpeI)||23.5||0µL||0.5µL||0µL||0µL||3µL||3µL||0µL||30µL | |1cut(SpeI)||23.5||0µL||0.5µL||0µL||0µL||3µL||3µL||0µL||30µL | ||
|- | |- | ||
- | |NC||02.3||0µL | + | |NC||02.3||0µL||0µL||0µL||0µL||1µL||1µL||5.7µL||10µL |
|} | |} | ||
Line 321: | Line 265: | ||
|2||RBS-lacZα-DT(XbaI&PstI) | |2||RBS-lacZα-DT(XbaI&PstI) | ||
|- | |- | ||
- | |3||RBS-lacZα-DT( | + | |3||RBS-lacZα-DT(NC) |
|} | |} | ||
- | [[File: | + | [[File:igku_91313.jpg]]<br> |
</div> | </div> | ||
Line 333: | Line 277: | ||
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2 | !state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2 | ||
|- | |- | ||
- | |experiment||9/13 Plac (SpeI&PstI)||8.5µL||9/13 pT181 antisense (XbaI&PstI)||2.1 µL|| | + | |experiment||9/13 Plac (SpeI&PstI)||8.5µL||9/13 pT181 antisense (XbaI&PstI)||2.1 µL||5.3 µL |
|- | |- | ||
- | |experiment||9/13 Plac (SpeI&PstI)||8.5 µL||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.8 µL|| | + | |experiment||9/13 Plac (SpeI&PstI)||8.5 µL||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.8 µL||5.15µL |
|- | |- | ||
- | |experiment||9/13 Plac (SpeI&PstI)||8.5 µL||9/2 pT181 attenuator(XbaI&PstI)||2.3 µL||10. | + | |experiment||9/13 Plac (SpeI&PstI)||8.5 µL||9/2 pT181 attenuator(XbaI&PstI)||2.3 µL||10.4 µL |
|- | |- | ||
- | |experiment||9/13 Plux (SpeI&PstI)||11.4 µL||9/13 RBS-lysis1-DT (XbaI&PstI)||5.0 µL|| | + | |experiment||9/13 Plux (SpeI&PstI)||11.4 µL||9/13 RBS-lysis1-DT (XbaI&PstI)||5.0 µL||8.2 µL |
|- | |- | ||
- | |experiment||9/13 Plux (SpeI&PstI)||11.4 µL||9/13 RBS-lysis2-DT (XbaI&PstI)||7.0 µL|| | + | |experiment||9/13 Plux (SpeI&PstI)||11.4 µL||9/13 RBS-lysis2-DT (XbaI&PstI)||7.0 µL||9.2 µL |
|- | |- | ||
- | |experiment||9/13 Plux (SpeI&PstI)||7.2 µL||9/13 RBS-lysis3-DT (XbaI&PstI)||6.0 µL|| | + | |experiment||9/13 Plux (SpeI&PstI)||7.2 µL||9/13 RBS-lysis3-DT (XbaI&PstI)||6.0 µL||6.6 µL |
|- | |- | ||
- | |experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 µL||9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)||5.2 µL|| | + | |experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 µL||9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)||5.2 µL||3.6 µL |
|- | |- | ||
- | |experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 µL||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.6 µL|| | + | |experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 µL||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.6 µL||1.8 µL |
|- | |- | ||
- | |experiment||9/12 Ptet-pT181 antisense (--)||1.0 µL||9/10 spinach-DT (--)||2.4 µL|| | + | |experiment||9/12 Ptet-pT181 antisense (--)||1.0 µL||9/10 spinach-DT (--)||2.4 µL||1.7 µL |
|} | |} | ||
Line 356: | Line 300: | ||
<span class="author">No name</span> | <span class="author">No name</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
- | !genome DNA|| | + | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total |
|- | |- | ||
- | | ||0.5||2.5||2.5||1.5||0.75||0.75|| || | + | |9/7 tetR aptamer 12_1R(pSB1C3) ||0.5||6.05||1.75||1.7||0.5||0||10.5 |
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/6 pT181 antisense-1(pSB1C3) ||0.5||6.05||1.75||1.7||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/6 pT181 attenuator (pSB1C3)||0.5||3.85||1.75||3.9||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |8/27 Pλ-RBS-luxI-DT-1||0.5||2.55||1.75||5.2||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |8/31 Pbad/araC-RBS-RFP-2||0.5||3.75||1.75||4||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/13 Plac(pSB1A2) ||0.5||4.2||1.75||3.5||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |8/20 J23100-RBS-lacZα-DT||0.5||6.35||1.75||1.4||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/7 Ptet-RBS-lacZα-DT||0.5||6.05||1.75||1.7||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/8 J23100-spinach-DT||0.5||5.75||1.75||2||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/11 RBS-lysis1-DT||0.5||6.05||1.75||1.7||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/12 RBS-lysis2-DT||0.5||6.75||1.75||1.0||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/12 RBS-lysis3-DT||0.5||6.75||1.75||1.0||0.5||0||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/8 J23100-spinach-DT||0.5||6.35||1.75||1.4||0||0.5||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |8/27 Pλ-RBS-luxI-DT-1||0.5||2.55||1.75||5.2||0||0.5||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/12 RBS-lysis2-DT||0.5||6.75||1.75||1.0||0||0.5||10.5 | ||
+ | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total | ||
+ | |- | ||
+ | |9/11 RBS-lysis1-DT||0.5||6.05||1.75||1.7||0||0.5||10.5 | ||
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
!PreDenature||Denature||Annealing||Extension||cycle | !PreDenature||Denature||Annealing||Extension||cycle | ||
|- | |- | ||
- | | | + | |96°C||96°C||50°C||68°C||-- |
|- | |- | ||
- | | | + | |2m||10s||5s||4m||30 |
|} | |} | ||
</div> | </div> | ||
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===PCR=== | ===PCR=== | ||
<div class="experiment"> | <div class="experiment"> | ||
- | <span class="author"> | + | <span class="author">Yoshida</span> |
{| class="wikitable" | {| class="wikitable" | ||
- | !genome DNA||KOD plus||10x buffer||dNTP||MgSO4|| | + | !genome DNA||KOD plus||10x buffer||dNTP||MgSO4||Prefix-RBS-RpaB_fwd primer||RpaB-GG4-suffix_rev primer||MilliQ||total |
|- | |- | ||
- | | ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25 | + | |RpaB||0.5||2.5||2.5||1.5||0.75||0.75||16.5||25 |
|} | |} | ||
+ | {| class="wikitable" | ||
+ | !genome DNA||KOD plus||10x buffer||dNTP||MgSO4||Prefix-PkaiBC_fwd primer ver.3||PkaiBC-suffix_rev primer ver.3||MilliQ||total | ||
+ | |- | ||
+ | |RpaB||0.5||2.5||2.5||1.5||0.75||0.75||16.5||25 | ||
+ | |} | ||
+ | |||
+ | |||
{| class="wikitable" | {| class="wikitable" | ||
!PreDenature||Denature||Annealing||Extension||cycle | !PreDenature||Denature||Annealing||Extension||cycle | ||
|- | |- | ||
- | | | + | |94°C||98°C||58°C||68°C||-- |
|- | |- | ||
- | | | + | |2m||10s||30s||30s||30 |
|} | |} | ||
</div> | </div> | ||
Line 403: | Line 429: | ||
|} | |} | ||
- | [[File: | + | [[File:igku_91318.jpg]]<br> |
- | [[File: | + | [[File:igku_91319.jpg]]<br> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
===Transformation=== | ===Transformation=== |
Latest revision as of 20:43, 27 September 2013
Contents |
Sep 13
Miniprep
DNA | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
9/12 Fusion6 antisense | 245.9 | 1.88 | 1.41 |
9/12 Plux-3 | 115.5 | 1.58 | 1.32 |
9/12 Plux-4 | 125.3 | 1.93 | 1.44 |
9/12 Fusion3n2 attenuator-1 | 231.9 | 1.81 | 1.31 |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kb ladder | -- |
2 | RBS-lysis1-DT | XbaI&PstI |
3 | RBS-lysis1-DT | XbaI&PstI |
4 | RBS-lysis1-DT | XbaI&PstI |
Lane | DNA | Enzyme |
---|---|---|
1 | 1kb ladder | -- |
3 | RBS-lysis2-DT | XbaI&PstI |
4 | RBS-lysis2-DT | XbaI&PstI |
5 | RBS-lysis2-DT | XbaI&PstI |
Lane | DNA | Enzyme |
---|---|---|
3 | RBS-lysis3-DT | XbaI&PstI |
4 | RBS-lysis3-DT | XbaI&PstI |
5 | RBS-lysis3-DT | XbaI&PstI |
6 | 1kb ladder | -- |
Lane | DNA | Enzyme |
---|---|---|
2 | 1kb ladder | -- |
3 | pSB1C3 | EcoRI&SpeI |
4 | pSB1C3 | EcoRI&SpeI |
5 | pSB1C3 | EcoRI&SpeI |
Lane | DNA | Enzyme |
---|---|---|
1 | 1kb ladder | -- |
2 | pSB1C3 | XbaI&PstI |
3 | pSB1C3 | XbaI&PstI |
4 | pSB1C3 | XbaI&PstI |
Restriction Enzyme Digestion
9/13 Plac | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cuts(PstI) | 16 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 7µL | 30µL |
NC | 0.8 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.2µL | 10µL |
9/10 Pcon attenuator | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cut(PstI) | 10 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 13µL | 30µL |
NC | 0.5 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
9/12 Plux | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cuts(PstI) | 12 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 11µL | 30µL |
NC | 0.6 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |
9/12 DT-E | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cut(XbaI) | 23.7 | 0µL | 0µL | 1µL | 0µL | 3µL | 3µL | 0µL | 30.7µL |
NC | 1.2 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.8µL | 10µL |
9/12 Pcon-RBS-luxR-bTE | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cuts(XbaI) | 22.4 | 0µL | 0µL | 1µL | 0µL | 3µL | 3µL | 0.6µL | 30µL |
NC | 2.2 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5.8µL | 10µL |
9/12 Pcon-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cut(SpeI) | 21 | 0µL | 1µL | 0µL | 0µL | 3µL | 3µL | 12.0µL | 30µL |
NC | 2.1 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5.7µL | 10µL |
M9 (EDTA)
1mM EDTA
volume | 10ml |
---|---|
Na2HPO4 | 300mg |
KH2PO4 | 150mg |
NaCl | 25mg |
NH4Cl | 50mg |
Fe(3)-EDTA | 21mg |
volume | 10ml |
---|---|
Na2HPO4 | 300mg |
KH2PO4 | 150mg |
NaCl | 25mg |
NH4Cl | 50mg |
Fe(3)-EDTA | 105mg |
</div>
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kb ladder | -- |
3 | Pcon-P | SpeI |
4 | Pcon-P | SpeI |
5 | Pcon-P | SpeI |
Liquid Culture
Sample | medium |
---|---|
9/11 aptamer12_1M(pSB1C3)-1 | -- |
9/11 aptamer 12_P1 | -- |
9/11 Fusaion1 antisense-4 | -- |
9/12 pSB4K5-1 | -- |
9/12 pSB4K5-2 | -- |
Restriction Enzyme Digestion
9/13 Plac-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cuts(SpeI) | 23.5 | 0µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |
NC | 8 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 0µL | 10µL |
9/13 Pcon-pT181 attenuator-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cut(SpeI) | 23.5 | 0µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |
NC | 02.3 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5.7µL | 10µL |
9/13 Plux-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
1cuts(SpeI) | 23.8 | 0µL | 0.2µL | 0µL | 1µL | 3µL | 3µL | 0µL | 30µL |
NC | 8 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 0µL | 10µL |
9/5 RBS-lacZα-DT | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total | |
---|---|---|---|---|---|---|---|---|---|
2cut(XbaI&PstI) | 10.8 | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 11.2µL | 30µL |
NC | 0.5 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
Electrophoresis
Ligation
state | Vector | Inserter | Ligation High ver.2 | ||
---|---|---|---|---|---|
experiment | 9/13 Plac (SpeI&PstI) | 8.5µL | 9/13 pT181 antisense (XbaI&PstI) | 2.1 µL | 5.3 µL |
experiment | 9/13 Plac (SpeI&PstI) | 8.5 µL | 9/10 aptamer 12_1R-DT (XbaI&PstI) | 1.8 µL | 5.15µL |
experiment | 9/13 Plac (SpeI&PstI) | 8.5 µL | 9/2 pT181 attenuator(XbaI&PstI) | 2.3 µL | 10.4 µL |
experiment | 9/13 Plux (SpeI&PstI) | 11.4 µL | 9/13 RBS-lysis1-DT (XbaI&PstI) | 5.0 µL | 8.2 µL |
experiment | 9/13 Plux (SpeI&PstI) | 11.4 µL | 9/13 RBS-lysis2-DT (XbaI&PstI) | 7.0 µL | 9.2 µL |
experiment | 9/13 Plux (SpeI&PstI) | 7.2 µL | 9/13 RBS-lysis3-DT (XbaI&PstI) | 6.0 µL | 6.6 µL |
experiment | 9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI) | 2.0 µL | 9/4 Plux-RBS-GFP-DT (EcoRI&SpeI) | 5.2 µL | 3.6 µL |
experiment | 9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI) | 2.0 µL | 9/10 aptamer 12_1R-DT (XbaI&PstI) | 1.6 µL | 1.8 µL |
experiment | 9/12 Ptet-pT181 antisense (--) | 1.0 µL | 9/10 spinach-DT (--) | 2.4 µL | 1.7 µL |
PCR
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/7 tetR aptamer 12_1R(pSB1C3) | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/6 pT181 antisense-1(pSB1C3) | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/6 pT181 attenuator (pSB1C3) | 0.5 | 3.85 | 1.75 | 3.9 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
8/27 Pλ-RBS-luxI-DT-1 | 0.5 | 2.55 | 1.75 | 5.2 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
8/31 Pbad/araC-RBS-RFP-2 | 0.5 | 3.75 | 1.75 | 4 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/13 Plac(pSB1A2) | 0.5 | 4.2 | 1.75 | 3.5 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
8/20 J23100-RBS-lacZα-DT | 0.5 | 6.35 | 1.75 | 1.4 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/7 Ptet-RBS-lacZα-DT | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/8 J23100-spinach-DT | 0.5 | 5.75 | 1.75 | 2 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/11 RBS-lysis1-DT | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/12 RBS-lysis2-DT | 0.5 | 6.75 | 1.75 | 1.0 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/12 RBS-lysis3-DT | 0.5 | 6.75 | 1.75 | 1.0 | 0.5 | 0 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/8 J23100-spinach-DT | 0.5 | 6.35 | 1.75 | 1.4 | 0 | 0.5 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
8/27 Pλ-RBS-luxI-DT-1 | 0.5 | 2.55 | 1.75 | 5.2 | 0 | 0.5 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/12 RBS-lysis2-DT | 0.5 | 6.75 | 1.75 | 1.0 | 0 | 0.5 | 10.5 |
genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA_fwd primer | SasA_rev primer | total |
---|---|---|---|---|---|---|---|
9/11 RBS-lysis1-DT | 0.5 | 6.05 | 1.75 | 1.7 | 0 | 0.5 | 10.5 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
96°C | 96°C | 50°C | 68°C | -- |
2m | 10s | 5s | 4m | 30 |
PCR
genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | Prefix-RBS-RpaB_fwd primer | RpaB-GG4-suffix_rev primer | MilliQ | total |
---|---|---|---|---|---|---|---|---|
RpaB | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |
genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | Prefix-PkaiBC_fwd primer ver.3 | PkaiBC-suffix_rev primer ver.3 | MilliQ | total |
---|---|---|---|---|---|---|---|---|
RpaB | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 58°C | 68°C | -- |
2m | 10s | 30s | 30s | 30 |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
3 | RBS-lacZα-DT | XbaI&PstI |
4 | RBS-lacZα-DT | XbaI&PstI |
5 | RBS-lacZα-DT | XbaI&PstI |
6 | RBS-lacZα-DT | XbaI&PstI |
Transformation
Name | Sample | Competent Cells | Total | Plate |
---|---|---|---|---|
9/13 Plac+pT181 antisense | 2µL | 20µL | 22µL | Amp |
9/13 Plac+apt12_1R | 2µL | 20µL | 22µL | Amp |
9/13 Plac+pT181 attenuator | 2µL | 20µL | 22µL | Amp |
9/13 Plux+pT181 antisense | 2µL | 20µL | 22µL | CP |
9/13 Plux+RBS-lysis2-DT | 2µL | 20µL | 22µL | CP |
9/13 Plux+RBS-lysis2-DT | 2µL | 20µL | 22µL | CP |
9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT | 2µL | 20µL | 22µL | Amp |
9/13 Pcon-pT181 antisense+apt12_1R-DT | 2µL | 20µL | 22µL | Amp |
9/13 Ptet-pT181antisense+spinach-DT | 2µL | 20µL | 22µL | CP |
Pλ-luxI-1 | 2µL | 20µL | 22µL | Amp |