Team:Evry/Notebook/w7

From 2012.igem.org

Weeks:

June July August September October November

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%

Wednesday, 25th July

Reception of primers fo Auxin Enzymes: IaaH FW and Rv and IaaM FW and RV.
PCR of imperial college BB with these primers to BB IaaH and IaaM. Electrophoresis show that results aren't good.

PCR:

PCR of pCS2+:
Reactants Volumes (µl)
GC Buffer 10
dNTPs 1
DNA 0,5
H2O 33
Primers (FW and RV) 2,5 each
fusion polymerase 0,5

PCR:

0,8% agarose

Thursday, 26th July

Test of auxin toxicity in tadpodes. Retry of IaaH and IaaM BB: Ok for IaaM but not for IaaH.

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