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Team:HokkaidoU Japan/Notebook/Promoter Construct Promoter Optimization Kit
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| - | <h2></h2> | + | <h2>Construct Promoter Optimization Kit</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">PCR & PCR purification</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Sep 12</time> </li> </ul> <div class="step-recipe"> vector:promoter(K1084001~010)-B0034-mRFP1-B0015-pSB1C3<br> V3-F, V1-R, KOD plus Neo / V3-F, V2-R, KOD plus Neo<br> Insert:R0040-B0034-lacZα-B0015<br> i1-F, i3-R, KOD plus Neo / i2-F, i3-R, KOD plus Neo </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 Sep 13</time> </li> </ul> <div class="step-recipe"> Cut V1:Promoter-βBsaI-BsaIψ-B0015-pSB1C3 with BsaI<br> Cut V2:promoter-B0034-γBsaI-BsaIψ-B0015-pSB1C3 with BsaI<br> Cut i1:βBsaI-R0040-B0034-lacZα-B0015-BsaIψ with BsaI<br> Cut i2:γBsaI-R0040-B0034-lacZα-B0015-BsaIψ with BsaI </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 Sep 14</time> </li> </ul> <div class="step-recipe"> Ligate V1 with i1, V1 with i2, V2 with i1, V2 with i2 </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 Sep 14</time> </li> </ul> <div class="step-recipe"> i1 on V1, i2 on V1, i1 on V2, i2 on V2<br> 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>Sun Sep 15</time> </li> </ul> <div class="step-recipe"> i1 on V1, i2 on V1, i1 on V2, i2 on V2 </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">PCR & PCR purification</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sun Sep 15</time> </li> </ul> <div class="step-recipe"> pormoter(K1084001~010)-B0034-lacZα-B0015<br> 100b up, 200b dn, KOD plus Neo </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>Sun Sep 15</time> </li> </ul> <div class="step-recipe"> Cut B0034-lacZα-B0015-pSB1AK3 with EcoRI, PstI(using 10xH buffer)<br> Cut R0040-B0034-lacZα-B0015 with EcoRI, PstI(using 10xH 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>Mon Sep 16</time> </li> </ul> <div class="step-recipe"> Ligate R0040-B0034-lacZα-B0015 with pSB1AK3 </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>Mon Sep 16</time> </li> </ul> <div class="step-recipe"> R0040-B0034-lacZα-B0015 on pSB1AK3<br> 5µL DNA to DH5α </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">PCR & PCR purification</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Mon Sep 16</time> </li> </ul> <div class="step-recipe"> aeBlue<br> gg-XbaI-F, 200b dn, KOD plus Neo </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 Sep 17</time> </li> </ul> <div class="step-recipe"> Cut R0010 with EcoRI, SpeI<br> Cut B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with XbaI, PstI<br> Cut R0010-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, PstI </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 Sep 17</time> </li> </ul> <div class="step-recipe"> Ligate R0010 with aeBlue/AmilCP/eforRed/AmilGFP, pSB1C3 </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>Tue Sep 17</time> </li> </ul> <div class="step-recipe"> R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 on pSB1C3<br> 5µL DNA to DH5α<br> </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">PCR & PCR purification</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Wed Sep 18, Thu Sep 19, Sat Sep 21</time> </li> </ul> <div class="step-recipe"> R0040-B0034-lacZα-B0015<br> i1-F, i3-R, KOD FX Neo / i2-F, i3-R, KOD FX Neo </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 Sep 19</time> </li> </ul> <div class="step-recipe"> Cut i1 with BsaI<br> Cut i2 with BsaI </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Sat Sep 21</time> </li> </ul> <div class="step-recipe"> i1, i2 </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 Sep 21</time> </li> </ul> <div class="step-recipe"> Cut i1 with BsaI<br> Cut i2 with BsaI </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>Sun Sep 22</time> </li> </ul> <div class="step-recipe"> Ligate V1 with i1, V1 with i2, V2 with i1, V2 with i2 </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>Sun Sep 22</time> </li> </ul> <div class="step-recipe"> i1 on V1, i2 on V1, i1 on V2, i2 on V2<br> 5µL DNA to DH5α </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>Mon Sep 23</time> </li> </ul> <div class="step-recipe"> i1 on V1, i2 on V1, i1 on V2, i2 on V2 </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>Mon Sep 23</time> </li> </ul> <div class="step-recipe"> R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015<br> 100b up, 200b dn, KOD FX Neo </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 Sep 24</time> </li> </ul> <div class="step-recipe"> Cut R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with XbaI, PstI(using 10xM buffer)<br> Cut i1 on V1(i2 on V1/i1 on V2/i2 on V2) with SpeI, PstI(using 10xH buffer) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Tue Sep 24</time> </li> </ul> <div class="step-recipe"> Digestion product </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 Sep 24</time> </li> </ul> <div class="step-recipe"> Ligate R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with [i1 on V1](i2 on V1/i1 on V2/i2 on V2) </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>Tue Sep 24, Wed Sep 25</time> </li> </ul> <div class="step-recipe"> R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) </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>Thu Sep 26</time> </li> </ul> <div class="step-recipe"> R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">PCR & Gel extraction</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Sep 26</time> </li> </ul> <div class="step-recipe"> E-X/S-P on pSB3K3<br> Km-gamma-f, Km-psy-r, KOD FX Neo </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Thu Sep 26</time> </li> </ul> <div class="step-recipe"> J31003 </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 Sep 27</time> </li> </ul> <div class="step-recipe"> Cut R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) with BsaI<br> Cut J31003 with BsaI </div> </div> </div> </li> | ||
| + | <li class="step"> <div class="step-tile"> <h2 class="step-title">EtOH precipitation</h2> </div> <div class="step-detail"> <div class="step-detail-body"> <ul class="step-detail-trials"> <li> <time>Fri Sep 27</time> </li> </ul> <div class="step-recipe"> Digestion product </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 Sep 27</time> </li> </ul> <div class="step-recipe"> Ligate R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) with J31003 </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 Sep 27</time> </li> </ul> <div class="step-recipe"> Ligation product<br> 5µL DNA to DH5α </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 Sep 27</time> </li> </ul> <div class="step-recipe"> Transformation product<br> to LBK plate </div> </div> </div> </li> | ||
<li class="step complete"> | <li class="step complete"> | ||
<div class="step-tile"><h2>Complete!</h2></div> | <div class="step-tile"><h2>Complete!</h2></div> | ||
Latest revision as of 07:09, 27 September 2013
Maestro E.coli
Notebook
Construct Promoter Optimization Kit
-
Start
-
PCR & PCR purification
vector:promoter(K1084001~010)-B0034-mRFP1-B0015-pSB1C3
V3-F, V1-R, KOD plus Neo / V3-F, V2-R, KOD plus Neo
Insert:R0040-B0034-lacZα-B0015
i1-F, i3-R, KOD plus Neo / i2-F, i3-R, KOD plus Neo -
Digestion
Cut V1:Promoter-βBsaI-BsaIψ-B0015-pSB1C3 with BsaI
Cut V2:promoter-B0034-γBsaI-BsaIψ-B0015-pSB1C3 with BsaI
Cut i1:βBsaI-R0040-B0034-lacZα-B0015-BsaIψ with BsaI
Cut i2:γBsaI-R0040-B0034-lacZα-B0015-BsaIψ with BsaI -
Ligation
Ligate V1 with i1, V1 with i2, V2 with i1, V2 with i2 -
Transformation
i1 on V1, i2 on V1, i1 on V2, i2 on V2
5µL DNA to DH5α -
Colony PCR
i1 on V1, i2 on V1, i1 on V2, i2 on V2 -
PCR & PCR purification
pormoter(K1084001~010)-B0034-lacZα-B0015
100b up, 200b dn, KOD plus Neo -
Digestion
Cut B0034-lacZα-B0015-pSB1AK3 with EcoRI, PstI(using 10xH buffer)
Cut R0040-B0034-lacZα-B0015 with EcoRI, PstI(using 10xH buffer) -
Ligation
Ligate R0040-B0034-lacZα-B0015 with pSB1AK3 -
Transformation
R0040-B0034-lacZα-B0015 on pSB1AK3
5µL DNA to DH5α -
PCR & PCR purification
aeBlue
gg-XbaI-F, 200b dn, KOD plus Neo -
Digestion
Cut R0010 with EcoRI, SpeI
Cut B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with XbaI, PstI
Cut R0010-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, PstI -
Ligation
Ligate R0010 with aeBlue/AmilCP/eforRed/AmilGFP, pSB1C3 -
Transformation
R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 on pSB1C3
5µL DNA to DH5α
-
PCR & PCR purification
R0040-B0034-lacZα-B0015
i1-F, i3-R, KOD FX Neo / i2-F, i3-R, KOD FX Neo -
Digestion
Cut i1 with BsaI
Cut i2 with BsaI -
EtOH precipitation
i1, i2 -
Digestion
Cut i1 with BsaI
Cut i2 with BsaI -
Ligation
Ligate V1 with i1, V1 with i2, V2 with i1, V2 with i2 -
Transformation
i1 on V1, i2 on V1, i1 on V2, i2 on V2
5µL DNA to DH5α -
Culture
i1 on V1, i2 on V1, i1 on V2, i2 on V2 -
PCR
R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015
100b up, 200b dn, KOD FX Neo -
Digestion
Cut R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with XbaI, PstI(using 10xM buffer)
Cut i1 on V1(i2 on V1/i1 on V2/i2 on V2) with SpeI, PstI(using 10xH buffer) -
EtOH precipitation
Digestion product -
Ligation
Ligate R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with [i1 on V1](i2 on V1/i1 on V2/i2 on V2) -
Transformation
R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) -
Mini-prep
R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) -
PCR & Gel extraction
E-X/S-P on pSB3K3
Km-gamma-f, Km-psy-r, KOD FX Neo -
EtOH precipitation
J31003 -
Digestion
Cut R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) with BsaI
Cut J31003 with BsaI -
EtOH precipitation
Digestion product -
Ligation
Ligate R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) with J31003 -
Transformation
Ligation product
5µL DNA to DH5α -
Culture
Transformation product
to LBK plate -
Complete!
