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Team:HokkaidoU Japan/Notebook/H Stem/Construction
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Revision as of 09:12, 14 October 2014
H-Stem Construction
-
Start
-
mini-Prep
1C3 Vector -
PCR
Plac (plasmid) with 100up-F & 200dn-R, Ptet-B0034-mRFP-dT (plasmid) with XhoI-RBS & NcoI-mRFP (anti-sense), double-Terminator (plasmid) with 100up & 200dn -
Digestion
Plac with SpeI, anti-sense with NcoI, anti-sense with XhoI, dT with XbaI -
Primer Annealing
stem-upstream-f & stem-upstream-r, stem-downstream-f & stem-downstream-r -
Ligation
Plac & stem-up & as-up, as-dn & stem-dn & dT -
PCR
Plac-stem-as with 100up-F & XhoI-RBS, as-stem-dT with NcoI-mRFP & 100dn -
PCR
Plac-stem-as & as-stem-dT with 100up-F & 200dn-R -
Digestion
Plac-stem-as-stem-dT with EcoRI & PstI, 1C3 with EcoRI & PstI -
Ligation
Plac-stem-as-stem-dT & 1C3 -
Transformation
Plac-stem-as-stem-dT on 1C3 -
Colony PCR
Plac-stem-as-stem-dT on 1C3 with stem-up-f & stem-d-r / 100up-F & 200dn-R / EX-F & PS-R -
Complete!
