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Team:HokkaidoU Japan/Notebook/H Stem/Construction
From 2014.igem.org
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li> | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li> | ||
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li> | ||
| - | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion# | + | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 02:48, 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 SpeI anti-sense with NcoI, XhoI B0015 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-up as-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 & PstI 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) 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℃ -
Complete!
