Template:IONIS Paris/Notebook/31-08-15
Digestion BBa_K1159001
| Tube | BBa_K1159001 |
|---|---|
| Buffer 2.1 | 2 μL |
| DNA | 10 μL |
| Enzyme EcoRI | 0,5 μL |
| Enzyme PstI | 0,5 μL |
37°C, 1h heat kill: 80°C, 20 min
Expected results
Results
BBa_K1159001: Not expected 1600 bp long band; the sequence of the part did not correspond to information published on the registry
Good amplification of Gblocks fragment (Holin/Endolysin, GFP, VVD YC and VVD YN)
No amplification of HOKD and CCDB fragment
Gel extraction (QIAgen kit)
PCR of Gblocks (CCDB and HOKD)
| Gblock | |
|---|---|
| MQ Water | 39 µL |
| RB Buffer | 5 µL |
| Mg2+ | 1,5 µL |
| dNTP 10 µM | 1 µL |
| Primer Fwd 25 µM | 0,5µL pSB1C3 Fwd |
| Primer Rev 25 µM | 0,5µL pSB1C3 Rev |
| DNA | 1µL |
| Taq Pol enzyme | 0,5 µL |
Expected results
Results
Good amplification gel extraction (QIAgen kit)
Liquid culture
2 colonies from culture on plates have been selected for amplification:
VVD YC/YN (x2)
VVD YC (x2)
VVD YN (x2)
GFP (x2)
Holin/Endolysin (x2)
CCDB (x2)
HOKD (x2)pDawn
- 20 ml LB + 20 µl Cam
pDusk
- 20 ml LB + 20 µl Kan
31 August 15