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

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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
PKU 20090707 Zhangs 1.JPG
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
PKU 20090707 Zhangs 2.JPG
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