August/20 August 2009

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1.digestion and ligation

digestion with restriction enzyme

K204007 , (K204003)

VectorInsert
RBS(1-2M)10cinR(1-14F)12
SpeI1XbaI1
PstI1PstI1
No.2 Buffer2No.22
dH2O6dH2O4
total20uLtotal20uL


K204008

VectorInsert
RBS(1-2M)10LuxI(2-8O)12
SpeI1XbaI1
PstI1PstI1
No.2 Buffer2No.22
dH2O6dH2O4
total20uLtotal20uL



37°C, 2hr


gel electrophoresis
gel cut

purification by [QIAquick Nucleotide Removal Kit]

ligation

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16°C overnight


2. rapid check

③(RBS+cinR),K204003

③-C,D : OK 

④(RBS+LasR),K204004

④-A,B,C : OK

⑥(RBS+cinI),K204006

⑥-A,B,C : OK