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Team:Evry/Notebook/w11
From 2013.igem.org
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| - | Chemically competent Top 10 <i>E. Coli</i> 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. | + | Chemically competent Top 10 <i>E. Coli</i> 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. |
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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 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. | ||
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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 | + | 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 got a 1 kb band which confirmed that we obtained the right amplificat. |
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Revision as of 14:43, 30 August 2013
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.
Integration
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 got a 1 kb band which confirmed that we obtained the right amplificat.
