IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Output/2009/06/29/Shan Shen

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1:30 Recycle the fragments of single digestion of T7 promoter.
Electrophoresis to recycle the fragments of 1-12M and 1-12O
The order and the amount of the samples: marker 10μL, plasmids of 1-12M 10μL; digestion products of 1-12M, plasmids of 1-12O, digestion products of 1-12O.
Results:
PKU 20090629 Shan Shen 1.JPG
2:00 Single digestion of the T7 promoter fragments

Fragments 10μL
Spe1 1.5μL
Buffer 2μL
ddH2O 5.5μL

2:20 Start to digest.
14:00 Electrophoresis to test the efficiency of double digestion
The order and the amount of the samples: marker 10μL, plasmids of T7 promoter 10μL, digestion products of T7 promoter 10μL.
Results:
PKU 20090629 Shan Shen 2.JPG
15:00 Recycle the fragments of T7 promoter, both double digestion and single digestion.
15:30 Add CIAP into another system of T7 promoter.
16:00 Electrophoresis to recycle the fragments of 1-12M and 1-12O
The order and the amount of the samples: marker 5μL, plasmids of T7 promoter 10μL, digestion products of T7 promoter 10μL.
16:50 Recycle the double digestion products of T7 promoter.
20:30 Ligation: T7 promoter + GFP

T7 promoter Double digestion with CIAP Double digestion without CIAP Single digestions separated
GFP 1-12M Medium rbs + GFP coding gene + terminator 1-12O Strong rbs + GFP coding gene + terminator

System of Ligation

7:1 (μL) 3:1 (μL) Control (μL)
Vector 1 1 1
Insert 7 3 0
T4 ligase 1 1 1
Buffer 1 1 1
ddH2O 0 4 7

21:00 Start to ligase