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Team:HokkaidoU Japan/Notebook/RBS SD8
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| - | <h2></h2> | + | <h2>SD8</h2> |
<div class="workflow-container"> | <div class="workflow-container"> | ||
<ul class="steps"> | <ul class="steps"> | ||
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<div class="step-tile"><h2>Start</h2></div> | <div class="step-tile"><h2>Start</h2></div> | ||
</li> | </li> | ||
| - | + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Primer Annealing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Feb 26</time> </li> </ul> <div class="step-recipe"> Anneal rbs-ensd-f and rbs-ensd-r<br> Anneal B0034-f and B0034-r </div> </div> </div> </li> | |
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Annealing two primers to synthesize our promoter.</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Feb 26</time> </li> </ul> <div class="step-recipe"> Anneal f primers and r primers </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Feb 26</time> </li> </ul> <div class="step-recipe"> plasmid extraction of R0040 on pSB1A2 and LacZα on pSB1AK2<br> However, we forgot to add RNAse to mP1. We failed in mini-prep. cry... </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Feb 26</time> </li> </ul> <div class="step-recipe"> B0015 100up, 200dn, KOD Plus NEO </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Feb 27</time> </li> </ul> <div class="step-recipe"> plasmid extraction of LacZα on pSB1AK2 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> Cut LacZ%alpha; SpeI and PstI(using 10×H buffer), B0015 fragment by XbaI and PstI(using 10×M buffer) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> Ligate RBS with R0040 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Gel Extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> LacZα </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ethanol Precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> LacZα </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Feb 28</time> </li> </ul> <div class="step-recipe"> R0040-RBS (SD8/ B0034) on pSB1A2<br> each of 1µL DNA to DH5α<br> faild </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 1</time> </li> </ul> <div class="step-recipe"> B0015(B0015) <br> faild </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 1</time> </li> </ul> <div class="step-recipe"> Cut B0015 XbaI and PstI (using 10×M buffer)@ </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 1</time> </li> </ul> <div class="step-recipe"> R0040-RBS (SD8/ B0034) on pSB1A2<br> each of 1µL DNA to JM109 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 1</time> </li> </ul> <div class="step-recipe"> R0040-(SD8,B0034) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 1</time> </li> </ul> <div class="step-recipe"> R0040-(SD8,B0034) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> Cut B0015(B0015) XbaI and PstI (using 10×M buffer) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> Ligate LacZαwith B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> LacZα-B0015 on pSB1A2<br> each of 1µL DNA to DH5α </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> R0040-SD8 in JM109 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> R0040-RBS(SD8x2) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-Prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 2</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034)<br> 100bup or 200bdn<br><br> Faild </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-Prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sun Mar 3</time> </li> </ul> <div class="step-recipe"> R0040-RBS(SD8) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sun Mar 3</time> </li> </ul> <div class="step-recipe"> LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sun Mar 3</time> </li> </ul> <div class="step-recipe"> LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-Prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Mar 4</time> </li> </ul> <div class="step-recipe"> LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Mar 4</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034)<br> 100bup or 200bdn<br> faild </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5</time> </li> </ul> <div class="step-recipe"> Cut R0040-RBS(SD8) SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using 10×M buffer) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Gel Extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5</time> </li> </ul> <div class="step-recipe"> R0040-SD8, LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ethanol Precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5</time> </li> </ul> <div class="step-recipe"> R0040-SD8, LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Liquid culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5</time> </li> </ul> <div class="step-recipe"> R0040-B0034 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034)<br> 100bup or 200bdn<br> faild </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Mar 5</time> </li> </ul> <div class="step-recipe"> Ligate R0040-RBS(SD8) with LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-Prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 6</time> </li> </ul> <div class="step-recipe"> R0040-B0034 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 6</time> </li> </ul> <div class="step-recipe"> R0040-RBS(SD8)-LacZα-B0015<br> each of 5µL DNA to DH5α </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Mar 7</time> </li> </ul> <div class="step-recipe"> LacZα-B0015-R0040-RBS(SD8) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Liquid culture</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Mar 7</time> </li> </ul> <div class="step-recipe"> R0040-SD8 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Digestion</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 8</time> </li> </ul> <div class="step-recipe"> Cut R0040-SD8 SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using10×M buffer) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Gel Extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 8</time> </li> </ul> <div class="step-recipe"> R0040-SD8 and LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ethanol Precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 8</time> </li> </ul> <div class="step-recipe"> R0040-SD8 and LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Ligation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 8</time> </li> </ul> <div class="step-recipe"> Ligate R0040-RBS(SD8) with LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Colony PCR</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 9</time> </li> </ul> <div class="step-recipe"> R0040-SD8-LacZα-B0015 on 1A2 in DH5α (yesterday we transformed again) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Transformation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 9</time> </li> </ul> <div class="step-recipe"> R0040-SD8-LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Mini-prep</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue mar 11</time> </li> </ul> <div class="step-recipe"> R0040-SD8-LacZα-B0015 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 12</time> </li> </ul> <div class="step-recipe"> R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">Sequencing</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 12</time> </li> </ul> <div class="step-recipe"> R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">β-galactosidase assay (1)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 12</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">β-galactosidase assay (2)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Mar 13</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">β-galactosidase assay (3)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Mar 14</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">β-galactosidase assay (4)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Mar 20</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">β-galactosidase assay (5)</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Mar 23</time> </li> </ul> <div class="step-recipe"> R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) </div> </div> </div> </li> | ||
<li class="step complete"> | <li class="step complete"> | ||
Revision as of 16:35, 27 September 2013
Maestro E.coli
Notebook
SD8
-
Start
-
Primer Annealing
Anneal rbs-ensd-f and rbs-ensd-r
Anneal B0034-f and B0034-r -
Annealing two primers to synthesize our promoter.
Anneal f primers and r primers -
Mini-prep
plasmid extraction of R0040 on pSB1A2 and LacZα on pSB1AK2
However, we forgot to add RNAse to mP1. We failed in mini-prep. cry... -
PCR
B0015 100up, 200dn, KOD Plus NEO -
Mini-prep
plasmid extraction of LacZα on pSB1AK2 -
Digestion
Cut LacZ%alpha; SpeI and PstI(using 10×H buffer), B0015 fragment by XbaI and PstI(using 10×M buffer) -
Ligation
Ligate RBS with R0040 -
Gel Extraction
LacZα -
Ethanol Precipitation
LacZα -
Transformation
R0040-RBS (SD8/ B0034) on pSB1A2
each of 1µL DNA to DH5α
faild -
Digestion
B0015(B0015)
faild -
Digestion
Cut B0015 XbaI and PstI (using 10×M buffer)@ -
Transformation
R0040-RBS (SD8/ B0034) on pSB1A2
each of 1µL DNA to JM109 -
Colony PCR
R0040-(SD8,B0034) -
Culture
R0040-(SD8,B0034) -
Digestion
Cut B0015(B0015) XbaI and PstI (using 10×M buffer) -
Ligation
Ligate LacZαwith B0015 -
Transformation
LacZα-B0015 on pSB1A2
each of 1µL DNA to DH5α -
Colony PCR
R0040-SD8 in JM109 -
Culture
R0040-RBS(SD8x2) -
Mini-Prep
R0040-RBS(B0034) -
Sequencing
R0040-RBS(B0034)
100bup or 200bdn
Faild -
Mini-Prep
R0040-RBS(SD8) -
Colony PCR
LacZα-B0015 -
Culture
LacZα-B0015 -
Mini-Prep
LacZα-B0015 -
Sequencing
R0040-RBS(B0034)
100bup or 200bdn
faild -
Digestion
Cut R0040-RBS(SD8) SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using 10×M buffer) -
Gel Extraction
R0040-SD8, LacZα-B0015 -
Ethanol Precipitation
R0040-SD8, LacZα-B0015 -
Liquid culture
R0040-B0034 -
Sequencing
R0040-RBS(B0034)
100bup or 200bdn
faild -
Ligation
Ligate R0040-RBS(SD8) with LacZα-B0015 -
Mini-Prep
R0040-B0034 -
Transformation
R0040-RBS(SD8)-LacZα-B0015
each of 5µL DNA to DH5α -
Colony PCR
LacZα-B0015-R0040-RBS(SD8) -
Liquid culture
R0040-SD8 -
Digestion
Cut R0040-SD8 SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using10×M buffer) -
Gel Extraction
R0040-SD8 and LacZα-B0015 -
Ethanol Precipitation
R0040-SD8 and LacZα-B0015 -
Ligation
Ligate R0040-RBS(SD8) with LacZα-B0015 -
Colony PCR
R0040-SD8-LacZα-B0015 on 1A2 in DH5α (yesterday we transformed again) -
Transformation
R0040-SD8-LacZα-B0015 -
Mini-prep
R0040-SD8-LacZα-B0015 -
Sequencing
R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn -
Sequencing
R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn -
β-galactosidase assay (1)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (2)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (3)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (4)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (5)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
Complete!
