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Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement
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Revision as of 14:59, 10 October 2014
Stem region replacement
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Start
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PCR
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pHN1257
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Transformation
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R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min
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Digestion
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Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer)
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Ligation
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Ligate R0010 with pHN1257
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Transformation
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Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min
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Colony PCR
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R0010-stem-dT 100up f 162primer R(あとで書く)
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mini-Prep
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R0010-stem-dT
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PCR(confirm)
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R0010-stem-dT 100up 162 KOD FX NEO
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Complete!
