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Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement
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Revision as of 11:26, 14 October 2014
Stem Region Replacement
-
Start
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PCR
pHN1257 161 Tm: 162 Tm: KOD FX NEO -
Transformation
R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Digestion
Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer) -
Ligation
Ligate R0010 with pHN1257 -
Transformation
Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Colony PCR
R0010-stem-dT 100up f 162primer R(あとで書く) -
mini-Prep
R0010-stem-dT -
PCR(confirm)
R0010-stem-dT 100up 162 KOD FX NEO -
Complete!
