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Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT
From 2014.igem.org
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| - | P<sub>tet</sub>-B0034-mRFP-dT(6A1) SpeI_PstI_F& P<sub>tet</sub>_SpeI_R(vector), | + | P<sub>tet</sub>-B0034-mRFP-dT(6A1) SpeI_PstI_F& P<sub>tet</sub>_SpeI_R(vector), XbaI_B0032_mRFP_F& PstI_SpeI_R(insert), KOD Fx Neo |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 13:32, 17 October 2014
hogehoge
Ptet-B0032-mRFP-dT
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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℃ 35min 65℃ 10min -
Ligation
Ligate vector with insert -
Transformation
ligation products on ice 30min each of 5.0µL DNA to DH5α 42℃ 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!
