Team:Evry/Notebook/w11

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<h1>Week 11: 26th August - 1st September</h1>
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<h1>Week 11: 26<sup>th</sup> August - 1<sup>st</sup> September</h1>
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<h2>TECAN</h2>
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<h2>Creation of a E. Coli &#916;Fur</h2>
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<h3>Strain preparation</h3>
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Chemically competent Top 10 <i>E. Coli</i> was transformed with the plasmid PTK which contained &#955; Red factor. We plated our bacteria on LB Agar with spectinomycin and let them grow overnight at 30°C.
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One colony had grown on our plate. We started an overnight culture of this colony in 2 mL of LB with spectinomycin and still at 30°C.
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We then diluted 1 mL of our culture in 100 mL of LB with spectinomycin and IPTG so the &#955; Red factor would start to be expressed. We let bacteria grew to a OD of 0.5 and made them electro competent. The protocol used can be found in our Protocol pages. 
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<h3>Homologous Recombination</h3>
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We realised a PCR on a plasmid with a kanamycin cassette resistance and with our two designed primers P119 and P120. The PCR product had been then purified and migrated on 1% agarose gel. We obtained the 1 kb band expected which confirmed that we got the right amplificat.
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We transformed by electroporation 50 uL of our strain with 100 ng of PCR product. We had a time constant of 5,6. After a 2 hours recovery in 2 mL of LB with IPTG, we added kanamycin and let our bacteria grow overnight at 30°C.
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Next day, our culture was very turbid. Our bacteria got a kanamycin resistance. We plated 50 uL of our culture on four kanamycin-agar petri dishes and grew overnight at 30°C.
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{{:Team:Evry/foot}}

Latest revision as of 15:22, 30 August 2013

Iron coli project

Week 11: 26th August - 1st September

TECAN

Creation of a E. Coli ΔFur

Strain preparation

Chemically competent Top 10 E. Coli was transformed with the plasmid PTK which contained λ Red factor. We plated our bacteria on LB Agar with spectinomycin and let them grow overnight at 30°C.

One colony had grown on our plate. We started an overnight culture of this colony in 2 mL of LB with spectinomycin and still at 30°C.

We then diluted 1 mL of our culture in 100 mL of LB with spectinomycin and IPTG so the λ Red factor would start to be expressed. We let bacteria grew to a OD of 0.5 and made them electro competent. The protocol used can be found in our Protocol pages.

Homologous Recombination

We realised a PCR on a plasmid with a kanamycin cassette resistance and with our two designed primers P119 and P120. The PCR product had been then purified and migrated on 1% agarose gel. We obtained the 1 kb band expected which confirmed that we got the right amplificat.

We transformed by electroporation 50 uL of our strain with 100 ng of PCR product. We had a time constant of 5,6. After a 2 hours recovery in 2 mL of LB with IPTG, we added kanamycin and let our bacteria grow overnight at 30°C.

Next day, our culture was very turbid. Our bacteria got a kanamycin resistance. We plated 50 uL of our culture on four kanamycin-agar petri dishes and grew overnight at 30°C.

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