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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%

Preparation of tadpole samples for HPLC

Aim: HPLC test to evaluate the presence of auxin in tadpoles embryos
Materiel:
- IAA powder (stocked at -20°C)
- NAA powder (stocked at room temperature)
- Acetonitrile 100% HPLC grade
- Methanol 100% HPLC grade
- Falcon 15ml
- 1,5ml tubes
- Pasteur pipettes
- Mortar
- Centrifuges
- Ice
- Vortex
- P1000, P200 with tips
The original protocol from Vastag L, Jorgensen P, Peshkin L, Wei R, Rabinowitz JD, et al. (2011) Remodeling of the Metabolome during Early Frog Development. PLoS ONE 6(2): e16881. doi:10.1371/journal.pone.0016881 article was performed on 1 egg of Xenopus laevis. Because 1 embryo of this species represents 10 embryos of Xenopus tropicalis, we decided to perform our protocol on 10 Xenopus tropicalis embryos.
Prerequisite: To have perform a FIV and the auxin toxicity protocol the day before.
Before beginning: Turn on the centrifuge at 4°C
1. Preparation of solutions (chemical hood)
- Mix for 10 samples
2ml acetonitrile
2ml methanol
1ml H2O
Cool mix to -20oC
- Preparation of auxin solutions 1mg/ml in 20% acetonitrile/H2O
NAA
1,8mg NAA
360ul acetonitrile
1440ul H2O
IAA
10,4mg IAA
2080ul acetonitrile
8320ul H2O
2. Preparation of the samples
Take 10 embryos of each condition in IAA and NAA (0, 125, 250 and 500 uM)
Rinse 4 times in H2O:
- Fill 4 wells of a 12 wells-plate with ddH2O
- Place the embryos of one condition in a well to another, to another…
Place the embryos in 1,5ml tubes
Add 55ul of cold mix
Crush the embryos with a mortar
Vortex
Place on ice for 20min
Centrifuge 5min – 14000 rpm – 4°C (centrifuge eppendorf 5417R)
Collect as much as possible of the supernatant and place it in new tubes
To extract more:
Add 55ul of the mix on the pellet
Repeat all steps

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.

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