Team:Paris Bettencourt/Notebook/TB-ception/Saturday 14th September.html

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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/TB-ception" target="_blank" class="tbnotelogo TClogo"> ASDF </a>
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<h3>Tuesday 10<sup>th</sup> July</h3>
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<h3>Saturday 14<sup>th</sup> September</h3>
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Fresh liquid cultures for: M smegmatis pMyC-GFP (kanamycine resistance) , M smegmatis, E.coli Bl21 carrying pET21b-TDMH and pColA-RFP (ampicilin and kanamycine resistances) and E.coli Bl21 carrying pET21b-TDMH, pColA RFP and pACyC-hly (ampicilin, kanamycine and chloramphenicol resistances)
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Fresh liquid cultures for :
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- M.smegmatis carrying pMyC-GFP(resistance kanamycine)
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<br />• CaCl2 competent cells for NEB Turbo - 25 mL of the culture (O.D. 1), 3 washing steps (1o min, 4 000 rpm, on 4.0). For the first one - 100 mM MgCl2, for the second - 100 mM CaCl 2, after the last one the cells were resuspended in CaCl2 + glycerol. I've also made electrocompetent cells (3 washes in dH20). 
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- M. smegmatis
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- E.coli Bl21 carrying pColA-RFP, pACYC-hly and pET21b-TDMH (resistance are respectively kanamycine, chloramphenicol and ampicilin). This liquid culture was made in the level 2 laboratory.
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<br />• cloning biobricked TDMH in the linear backbone - protocol from the 12th of September. New competent cells and new Chl plates.
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<br />• pET+pSB3C5 worked - miniprep; repeated the Gibson for pACYC
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<br />• PCR for confirming pET+pSB3C5 - tamplet is the miniprep and the primers are pDCo4 R/F. pET Duet as the positive controle (Phusion protocol).

Latest revision as of 14:43, 25 September 2013

TB-ception

Saturday 14th September


Fresh liquid cultures for: M smegmatis pMyC-GFP (kanamycine resistance) , M smegmatis, E.coli Bl21 carrying pET21b-TDMH and pColA-RFP (ampicilin and kanamycine resistances) and E.coli Bl21 carrying pET21b-TDMH, pColA RFP and pACyC-hly (ampicilin, kanamycine and chloramphenicol resistances)

• CaCl2 competent cells for NEB Turbo - 25 mL of the culture (O.D. 1), 3 washing steps (1o min, 4 000 rpm, on 4.0). For the first one - 100 mM MgCl2, for the second - 100 mM CaCl 2, after the last one the cells were resuspended in CaCl2 + glycerol. I've also made electrocompetent cells (3 washes in dH20).

• cloning biobricked TDMH in the linear backbone - protocol from the 12th of September. New competent cells and new Chl plates.

• pET+pSB3C5 worked - miniprep; repeated the Gibson for pACYC

• PCR for confirming pET+pSB3C5 - tamplet is the miniprep and the primers are pDCo4 R/F. pET Duet as the positive controle (Phusion protocol).

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