Template:IONIS Paris/Notebook/02-09-15
Digestion of miniprep from 01/09 and BBa_K1159001
| Tube | Miniprep (01/09) | BBa_K1159001 |
|---|---|---|
| MQ Water | 12 µL | 12 µL |
| Buffer 2.1 | 2 µL | 2 µL |
| DNA | 5 µL | 5 µL |
| EcoRI | 0,5 µL | 0,5 µL |
| PstI | - | 0,5 µL |
| BamHI | - | 0,5 µL |
37°C, 1h
Expected results
Results
Interesting band for Construction with VVD YC and VVD YN, require control test;
BBa_K1159001: we get 2 on 3 expected bands, confirmation of the structure of the plasmid with a pBad promoter (not indicated on the registry) before the nanoluc gene.
Liquid culture
pDawn – H/E 20 mL LB + 20 µL Kan, without light;
pDawn – GFP 20 mL LB + 20 µL Kan, with light;
pDawn – HOKD 20 mL LB + 20 µL Kan, without light;
pDawn – CCDB 20 mL LB + 20 µL Kan, without light;
pSB1C3– CCDB;
pSB1C3 – HOKD;
pDawn Gblock I-V purification
10 µl of ligation product + 40 µl of H2O MQ;
Add 1/10 (5µL) of the volume of NaOAc 3M pH 5.2;
Add 2,5 volumes (137,5 µl) of Ethanol 100%;
Mix and stored at -20°C for 1 hour at least;
Centrifuge 30 sec, 14000 rpm at 4°C;
Discard the supernatant;
Resuspend the pellet with 500 µl of ethanol 70%, invert up the tube several time;
Centrifuge 5 min, 14000 rpm at 4°C;
Discard the supernatant and let dry the pellet;
Resuspend the pellet with the right amount of water following the final concentration expected (10µL here)
2 September 15