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Team:HokkaidoU Japan/Notebook/H Stem/Construction
From 2014.igem.org
(Difference between revisions)
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<div class="step-recipe"> | <div class="step-recipe"> | ||
R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ | R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃ | R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃ | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ | B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ | ||
| - | |||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
R0010 with SpeI | R0010 with SpeI | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
anti-sense with NcoI, XhoI | anti-sense with NcoI, XhoI | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
B0015 with XbaI | B0015 with XbaI | ||
| - | |||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃ | stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃ | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | stem_downstream_F Tm:71.4℃ & stem_downstream_R Tm:71.4℃ | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
R0010 & stem-up & as-up | R0010 & stem-up & as-up | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | as-dn & stem-dn & B0015 | ||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | + | R0010-stem-as with 100up_F Tm:62.6℃ & XhoI_RBS_R Tm:68.2℃ | |
| - | + | </div> | |
| + | <div class="step-recipe"> | ||
| + | as-stem-B0015 with NcoI-mRFP_F Tm:76.0℃ & 200dn Tm:66.6℃ | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
Cut R0010-stem-as-stem-B0015 with EcoRI & PstI | Cut R0010-stem-as-stem-B0015 with EcoRI & PstI | ||
| - | + | </div> | |
| + | <div class="step-recipe"> | ||
| + | Cut pSB1C3 with EcoRI & PstI | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
R0010-stem-as-stem-B0015(on pSB1C3) | R0010-stem-as-stem-B0015(on pSB1C3) | ||
| - | + | </div> | |
| + | <div class="step-recipe"> | ||
| + | 5.0µL to DH5alpha; | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | R0010-stem-as-stem-B0015(on pSB1C3) with stem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃ / 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ / EX_F Tm:70.4℃ & PS_R Tm:74.6℃ | + | R0010-stem-as-stem-B0015(on pSB1C3) with |
| - | + | </div> | |
| + | <div class="step-recipe"> | ||
| + | stem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃ | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | EX_F Tm:70.4℃ & PS_R Tm:74.6℃ | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 03:29, 18 October 2014
Notebook
Lab Documents
H-Stem Construction
-
Start
-
Mini-Prep
pSB1C3 vector -
PCR
R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ -
Digestion
R0010 with SpeIanti-sense with NcoI, XhoIB0015 with XbaI -
Primer Annealing
stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃stem_downstream_F Tm:71.4℃ & stem_downstream_R Tm:71.4℃ -
Ligation
R0010 & stem-up & as-upas-dn & stem-dn & B0015 -
PCR
R0010-stem-as with 100up_F Tm:62.6℃ & XhoI_RBS_R Tm:68.2℃as-stem-B0015 with NcoI-mRFP_F Tm:76.0℃ & 200dn Tm:66.6℃ -
PCR
R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃ -
Digestion
Cut R0010-stem-as-stem-B0015 with EcoRI & PstICut pSB1C3 with EcoRI & PstI -
Ligation
Ligate R0010-stem-as-stem-B0015 with pSB1C3 -
Transformation
R0010-stem-as-stem-B0015(on pSB1C3)5.0µL to DH5alpha; -
Colony PCR
R0010-stem-as-stem-B0015(on pSB1C3) withstem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃EX_F Tm:70.4℃ & PS_R Tm:74.6℃ -
Complete!
