IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Output/2009/07/07/Zhangs

Identify the RBS+CI+T ligation product by electrophoresis
Result (from left to right)
2K+CI+1-23L①~⑤ (plasmid), 2M+CI+1-23L①~⑤ (plasmid), 1J+CI_1-23L①~⑤ (plasmid), RBS plasmid control, Marker

The 1-2K+CI+T1, 2, 3, 5 & 1-2M+CI+T1, 2, 3, 4, 5 & 1-1J+CI+T1, 2, 4, 5 are target plasmids.
Choose samples:
[1-1H⑤,1-2G⑤,1-5N⑤, 1-2I⑤, 1-11N④, 1-2K⑤, 1-2M⑤, 1-1J④]+CI+1-23L, 8 samples in total.
Double-digestion of RBS+CI+1-23L insert

1.5μL	XbaI
1.5μL	PstI
2μL	10×M buffer
10μL	Plasmid
5μL	ddH2O
20μL	Total

Electrophoresis
Result (from left to right)
1-2K, 1-2M, 1-1H, 1-11N, 1-2G, 1-2I, 1-5N, 1-1J, Plasmid Control, Marker

1-2K+CI+1-23L insert digesting failed

Gel Extraction for RBS+CI+1-23L

Gel		PN volume
2M	0.037g	111μL
1H	0.027g	81μL
11N	0.018g	54μL
2G	0.036g	108μL
2I	0.028g	84μL
5N	0.039g	117μL
1J	0.045g	135μL

Ligate the RBS+CI+1-23L insert and T7 promoter vector
System

3μL	insert
1μL	vector
1μL	10× Ligase buffer
1μL	Ligase
4μL	ddH2O
10μL	Total