August/20 August 2009

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1.digestion and ligation

digestion with restriction enzyme

K204007 , (K204003)

Vector		Insert
RBS([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 1-2M])	10	cinR([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F])	12
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2 Buffer	2	No.2	2
dH₂O	6	dH₂O	4
total	20uL	total	20uL

K204008

Vector		Insert
RBS([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 1-2M])	10	LuxI([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 2-8O])	12
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2 Buffer	2	No.2	2
dH₂O	6	dH₂O	4
total	20uL	total	20uL

↓
37°C, 2hr

↓
gel electrophoresis

UP   : 1-2M , 1-14F , 1-2M , 2-8O
DOWN : No.3-A,B,C,D , No.4-A,B,C,D , No.6-A,B,C,D , Vector

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