IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/09/20: Difference between revisions

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**pSB1A2::R0011 (pLac) + pSB1C3::supD
**pSB1A2::R0011 (pLac) + pSB1C3::supD


I will to perform double transformation for this.
I will need to perform double transformation for this.
I transform 1.5µL of miniprep of the above plasmids, in 20µL of MG1655 competent cell.
I transform 1.5µL of miniprep of the above plasmids, in 20µL of MG1655 competent cell.
I plate 200µL after 1h45 of recovery on Cm+Amp, expect for the first double transformation, where i plate on Kan+Cm+Amp.
I plate 200µL after 1h45 of recovery on Cm+Amp, except for the first double transformation, where i plate on Kan+Cm+Amp.




Revision as of 10:59, 22 October 2012

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Result Transformation

It works weirdly, because there are colonies on positive control, none on negative control, and there is an absence of colonies in the middle of the ligation transformation plates, that can be explain by the platin on Cm on plain LBA plates, and we would have a gradient of antibiotic.

So I took 2 colonies from each plate, the ones closer to the hole, and i make them grow in LB+Cm.The culture tube are composed of 5mL of LB + 5µL of Cm.

I also made a PCR colony with the same colonies.

Here is the content of 1 tube of PCR :

  • VR (diluted 10 times from the stock tube) : 2,5µL
  • VF2 (diluted 10 times from the stock tube) : 2,5µL
  • PCR MM 2X : 25 µL
  • water : 20 µL

Here is the PCR pgm :

  • 95°C - 15' (to lyse cells too)
  • 95°C - 30"
  • 50°C - 30"
  • 72°C - 40"
  • 72°C - 10'
  • 4°C -[math]\displaystyle{ \infty }[/math]

-> 25 cycles

  • Result of PCR on colonies :
Agarose gel after PCR on colonies after transfomrtion of ligation product. Tha bad oare expected : P1003 is about 1000bp in length, and supD+T is a bit more that 200 bp.

Since the PCR is good, the sequence should be good too. We can register 2 new biobricks, and wait for the sequencing results


Amp 7*

My only clone from yesterday grew up in LB+Amp, and I miniprep it and transform it in either NEB turbo or in MG1655 with or without the other plasmid that contain supD

Result QCM

I get 2 more colonies on Amp and 3 on Tet -> liquid culture.

I also run a gel with the product of Digestion before transformation.

Agarose gel after PCR site directed mutagenesis and Digestion with DpnI. There are 2 samples for each plasmid modified, and W is for water, when the PCR was running without plasmids, only primers. We can see a band for plasmid pSB1A2 and plasmid pSB4K5

The band corresponding to pSB1A1 can explain why we have colonies on Amp plates, but I still don't have colonies on Kan plates, and I have colonies on TetR, it might be contaminants here. Further experiments will make that clear.

I will miniprep the liquid culture of this morning, and then transform them in MG1655 with or without pSB1C3::supD.

  • So I minipreped the 5 cultures, and it turns out that I had no plasmid DNA for all 3 colonies from Tet plates (3ng/µL in Nanodrop), meanings that these clones are contaminants, or became resistant to Tetracycline meanwhile, which is unlikely.

I transformed then only the 3a, 3b, and I did 7 again. It is not exactly the same process : I co-transform alway either pSB1C3::supD or pSB1C3::RFP, that give 6 different transformations. 12 actually since I made duplicate.

1.5µL of plasmid in 20µL of cell. Heat shock as usual, 1h45 of recovery. 200µL plated on Cm + Amp.

Preparation of TECAN experiment

  • Here we want to quantify the leakiness of one amber codon. For that we will set up an growth experiment overnight, wi the Tecan, that will be able measure OD600 all night long. We will have 4 differents strains, all in MG1655 with:
    • pSB1A1::P1003 (KanR - pSC009) + pSB1C3::supD (pSC011) : positive control
    • pSB1A1::P1003* (Kan* - pSC012 (ie with an amber codon))
    • pSB1A1::P1003* + pSB1C3::supD
    • pSB1A2::R0011 (pLac) + pSB1C3::supD

I will need to perform double transformation for this. I transform 1.5µL of miniprep of the above plasmids, in 20µL of MG1655 competent cell. I plate 200µL after 1h45 of recovery on Cm+Amp, except for the first double transformation, where i plate on Kan+Cm+Amp.


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