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Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP
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Revision as of 07:09, 25 September 2014
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Start
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Primer Annealing
asB0034-f Tm: 68.7°C asB0034-r Tm: 68.7°C -
Digestion
Cut pHN1257 with NcoI, XhoI(using 10×cut smart) -
Gel extraction
digested pHN1257 -
Ethanol Precipitation
digested pHN1257 -
Ligation
Ligate pHN1257 with asB0034 -
Transformation
Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Colony PCR
Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1 asB0034-R Tm:68.7 -
Culture
Ptrc-stem-asB0034-stem -
mini-Prep
Ptrc-stem-asB0034-stem -
Transformation
Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice 30min each of 2.5µL DNA to DH5α 42C 30sec on ice 2min -
Complete!
