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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