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Revision as of 16:04, 5 August 2012 (view source) Chr.karine (Talk | contribs) ← Older edit		Latest revision as of 10:01, 9 August 2012 (view source) Chr.karine (Talk | contribs)
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	Gel 0,8%		Gel 0,8%
-	<h3>Preparation of tadpole samples for HPLC</h3>
-	<b>Aim:</b> HPLC test to evaluate the presence of auxin in tadpoles embryos<br>
-	<b>Materiel:</b><br>
-	- IAA powder (stocked at -20°C)<br>
-	- NAA powder (stocked at room temperature)<br>
-	- Acetonitrile 100% HPLC grade<br>
-	- Methanol 100% HPLC grade<br>
-	- Falcon 15ml<br>
-	- 1,5ml tubes<br>
-	- Pasteur pipettes<br>
-	- Mortar<br>
-	- Centrifuges<br>
-	- Ice<br>
-	- Vortex<br>
-	- P1000, P200 with tips<br>
-	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.<br>
-	<b>Prerequisite:</b> To have perform a FIV and the auxin toxicity protocol the day before.<br>
-	<b>Before beginning:</b> Turn on the centrifuge at 4°C<br>
-	<ul>
-	<li><b>1.Preparation of solutions (chemical hood)</b><br>
-	</ul>
-	<b>Mix for 10 samples</b><br>
-	2ml acetonitrile<br>
-	2ml methanol<br>
-	1ml H2O<br>
-	Cool mix to -20oC<br>
-	<b>Preparation of auxin solutions 1mg/ml in 20% acetonitrile/H2O</b><br>
-	NAA<br>
-	1,8mg NAA<br>
-	360ul acetonitrile<br>
-	1440ul H2O<br>
-	IAA<br>
-	10,4mg IAA<br>
-	2080ul acetonitrile<br>
-	8320ul H2O<br>
-	<ul>
-	<li><b>Preparation of the samples</b><br>
-	</ul>
-	Take 10 embryos of each condition in IAA and NAA (0, 125, 250 and 500 uM)<br>
-	Rinse 4 times in H2O:<br>
-	- Fill  4 wells of a 12 wells-plate with ddH2O<br>
-	- Place the embryos of one condition in a well to another, to another…<br>
-	Place the embryos in 1,5ml tubes<br>
-	Add 55ul of cold mix<br>
-	Crush the embryos with a mortar <br>
-	Vortex<br>
-	Place on ice for 20min<br>
-	Centrifuge 5min – 14000 rpm – 4°C (centrifuge eppendorf 5417R)<br>
-	Collect as much as possible of the supernatant and place it in new tubes<br>
-	<b>To extract more:</b><br>
-	Add 55ul of the mix on the pellet<br>
-	Repeat all steps<br>

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Latest revision as of 10:01, 9 August 2012

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