Team:Paris Saclay/Notebook/September/4

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(Lab Work)
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I use the same PCR condition than [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/2#Limonene_synthase September 2] but with the right primer and plasmid
I use the same PCR condition than [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/2#Limonene_synthase September 2] but with the right primer and plasmid
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[[File:0309 Vérif PCR GS CAD PS LS Chromo.jpg|400px]]
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[[File:0309 Vérif PCR GS CAD PS LS Chromo.jpg|400px|center]]
We can see that we don't have any insert in our plasmid
We can see that we don't have any insert in our plasmid
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We find some other PCR of BBa K762100 and  GS and  BBa_K517003 so we check it by electrophoresis
We find some other PCR of BBa K762100 and  GS and  BBa_K517003 so we check it by electrophoresis
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[[File:0309 Vérif PCR GS CAD PS LS Chromo.jpg|400px]]
+
[[File:0309 Vérif PCR GS CAD PS LS Chromo.jpg|400px|center]]
And I purify the PCR
And I purify the PCR
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[[File:0309 PCR cleanup GS PS CAD.jpg|400px]]
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[[File:0309 PCR cleanup GS PS CAD.jpg|400px|center]]
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Transformation with competent bacteria
Transformation with competent bacteria
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add DNA (Top vector our pPSII)
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add DNA (Top vector)
30' on ice
30' on ice

Latest revision as of 12:52, 14 October 2014

Contents

Thursday 4st September

Lab Work

By Mélanie

Lemon Scent

Verfication of the pPS3/4/5 plasmid

Due to the strange results obtained last day, I do a PCR of the insert with the differents plasmid : I use the same PCR condition than September 2 but with the right primer and plasmid

0309 Vérif PCR GS CAD PS LS Chromo.jpg

We can see that we don't have any insert in our plasmid

PCR verification

We find some other PCR of BBa K762100 and GS and BBa_K517003 so we check it by electrophoresis

0309 Vérif PCR GS CAD PS LS Chromo.jpg

And I purify the PCR

0309 PCR cleanup GS PS CAD.jpg


Well 1 = Ladder Well 2 = GS Well 3 = PS Well 4 = CAD

We can see that we don't have lost a lot of our PCR product

cloning

Cloning of PCR purification in a TA plasmid (Topo plasmid)

First I had to add AAA to the PCR

component volume
buffer 1μl
dATP 1μl
PCR 7.5μl
Taq 0.5μl

72° 15'

and

component volume
H2O 1μl
buffer 1μl
vector 1μl
cloning 1μl

30' at room temperature

Transformation

Transformation with competent bacteria

add DNA (Top vector)

30' on ice 45' 42°c 30' at 37°

spread on petri dish

Photo of the Day

Paris Saclay 4 september.jpg

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