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