From 2012.igem.org
Weeks
Week 7: 23rd July - 29th July
Monday, 23rd July
Cloning:
4 different clones pCS2+ RFP => incubation in LB medium overnight at 37 degree celsius.
PCR:
PCR of pCS2+:
| Reactants |
Volumes (µl) |
| GC Buffer |
10 |
| dNTPs |
1 |
| DNA |
1 |
| H2O |
32,5 |
| Primers (FW and RV) |
2,5 each |
| fusion polymerase |
0,5 |
Xenopus
Start of auxin's toxicity test on tadpoles: Tadpoles in there growth media + 0/125/250 or 500 µM auxin
''We changed this media each days during one week''
Tuesday, 24th July
Plasmid Purification:
On the pCS2+ RFP clones incubated the day before:
DNA Concentration
| Tube |
Concentration (ng.uL-1) |
| 1 |
144,2 |
| 2 |
161,9 |
| 3 |
184,9 |
| 4 |
120,7 |
Digestion:
Plasmid digestion
| Tube |
V DNA (uL) |
V SpeI (uL) |
V EcoRI (uL) |
V buffer (uL) |
V H2O (uL) |
| 1 |
7 |
1 |
1 |
2 |
9 |
| 2 |
6,2 |
1 |
1 |
2 |
9,8 |
| 3 |
5,4 |
1 |
1 |
2 |
10,6 |
| 4 |
8,3 |
1 |
1 |
2 |
7,7 |
Gel migration:
Gel 0,8%
Thursday, 26th July
Test of auxin toxicity in tadpodes.
Friday, 27th July
- PCR of TirI: One PCR with primers TirI FW + Sdm RV / One PCR with primers TirI RV + Sdm FW
- Gel extraction of these PCR
- New PCR with the mix of the two PCR products + primers TirI FW and RV
"
