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From 2013.igem.org

Revision as of 09:43, 11 September 2013 (view source) Zyi (Talk | contribs) (→Lab work) ← Older edit		Revision as of 14:01, 22 September 2013 (view source) Nad' (Talk | contribs) Newer edit →
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-	{{Team:Paris_Saclay/incl_debut_generique}}
-	='''Notebook : July 3'''=
-
-	=='''Summary:'''==
-	FNR regulator system:
-
-	*Continued what we started yesterday. Observed the Petri dish, selected the colonies. 4 colonies in total, they were 2 include FNR+plasmid in PSB1C3 and 2 from the control.
-	*Used 4 primers: VF2, VR, Pfnr_up, Pfnr_down for the verification test. They were designed to cut 4 special sites for creating 3 different regions on plasmid chain: VF2/VR, VF2/PFNR_down, PFNR_up/VR. After the amplification, those PCR products had been put on electrophoresis gel for the verification.
-
-	=='''Lab work'''==
-
-	*A.aero/anaerobic regulation system
-	:*2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
-	:*BioBrick RBS+amilCP+terminator in plasmid PSB1C3
-
-	<u>Observation</u>
-
-	<p>After the incubation overnight, we observed the Petri dishes. </p>
-	<br>
	{| border="1" align="center"		{| border="1" align="center"
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		+
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		+
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		+	{{Team:Paris_Saclay/incl_debut_generique}}
		+
		+	='''Notebook : July 1'''=
		+
		+	=='''Lab work'''==
		+
		+	==='''A - Aerobic/Anaerobic regulation system'''===
		+
		+	===='''Objective : obtaining Bba_K1155000'''====
		+
		+	===='''1 - Colony PCR of Bba_K1155000, Bba_I732017, Bba_K592009'''====
		+
		+	Abdou, Sheng, Zhou
		+
		+	{|
		+	| style="border:1px solid black;padding:5px;background-color:#DE;" |
		+	Tranformation of 07/02/13 works. We will do a Colony PCR of colonies.
		+	|}
		+
		+	Colonies count :
		+
		+	* Standard concentration :
		+	** Bba_K1155000 : 0
		+
		+	* High concentration :
		+	** Bba_K1155000 : 2
		+
		+	<u>Primer and PCR</u>
		+
		+	<p>VF2, VR, Pfnr_up, Pfnr_down are four oligos that we used for plasmid amplification. We used tree combinaisons VF/VR, VF/Pfnr_Down, Pfnr_Up/VR.
		+	If the promoter Pfnr insert PSB1C3 plasmid successfully, tree fragments with specific size will be amplified. </p><br>
		+	<center>[[File:PSprimer07.jpg|300px]]</center>
	<br>		<br>
		+
		+
	{| border="1" align="center"		{| border="1" align="center"
-	|[[Team:Paris Saclay/Notebook/July/2|<big>Previous day</big>]]	+	|[[Team:Paris Saclay/Notebook/June/30|<big>Previous day</big>]]
	|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]		|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
-	|[[Team:Paris Saclay/Notebook/July/4|<big>Next day</big>]]	+	|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</big>]]
	|}		|}
	{{Team:Paris_Saclay/incl_fin}}		{{Team:Paris_Saclay/incl_fin}}

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Revision as of 14:01, 22 September 2013

colonies	Normal concentration	High concentration
control	11	60
Fnr in plasmid PSB1C3	0	2

We picked up 4 colonies for further test (2 include FNR+plasmid in PSB1C3 and 2 from the control)

Primer and PCR

VF2, VR, Pfnr_up, Pfnr_down are 4 primes that we used for plasmid restriction. The primers, if the promoter fnr entre the plasmid successfully will amplify 3 sub-pieces with specific size. They are VF/VR, VF/Pfnr_down, Pfnr_up/VR.

So we had prepared 4(colonies)*3(amplification) = 12 PCR tubes.

• Dream Taq(5µg/µl):2µl
  
• Buffer (Dream Taq) 10X:10µl
  
• dNTP:2µl
  
• Primer (F/R;F/fnr_R;fnr_F/R):2µl+2µl
  
• H2O:82µl
  
• Total:100µl (volume for 4 tubes, so 25µl for each)
  

PCR programe

The PCR products was put on a gel of agarose 1.5% with BET (1.5%), migrated during 30 min at 100V. the picture analysis was be delayed to the next day.

Culture confirmation

We performed another colonies seeding. 2 colonies selected from each Petri dish were seeded in liquid medium with their corresponding antibiotic at 37°C under stirring during 1 night.