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Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT
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Revision as of 09:43, 25 September 2014
P-tet-B0032-mRFP-pdT
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Start
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PCR
Ptet-B0034-mRFP-dT(6A1) SpeI-PstI-F& Ptet-SpeI-R(vector), XbaI-B0032-mRFP-F& PstI-SpeI-R(insert), KOD Fx Neo -
Gel extraction
digested PCR productions -
Digestion
Cut vector with SpeI, PstI (using 10×H buffer) Cut insert with XbaI, PstI (using 10×M buffer) -
Antarctic phosphatase
Antactic phosphorylate insert (using 10×Antarctic phosphatase buffer) 37°C 35min 65°C 10min -
Ligation
Ligate vector with insert -
Transformation
ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Colony PCR
Ptet-B0034-mRFP-dT on pSB6A1 EcoI-XbaI-F Tm:70.9 rbs-SpeI-R Tm:59.2 -
Culture
Ptet-B0034-mRFP-dT on pSB6A1 -
mini-Prep
Ptet-B0034-mRFP-dT on pSB6A1 -
Complete!
