Team:Paris Liliane Bettencourt/Notebook/2011/07/26/

From 2011.igem.org

Team IGEM Paris 2011

Cyrille

The miniprep was made on the morning by Axel. In the mean time, some glycerols where made from the CodY- strains sended by Linc Sonenshein, and grown overnight.

The plasmid extracted was loaded on a gel. The plasmid alone, the plasmid digested during 5 min by a fast digest EcoRI, and digested one hour with HincII and XmnI.

The result are shown below:

DNA ladder - Plasmid pHM3 from the miniprep - Plasmid pHM3 digested by EcoRI - Plasmid digested by HincII and XmnI

The conclusion is that this time, we have the good plamid and this time it is visible on the gel.

The QCM was launched again, with the new DNA template, and launched overnight. A few cycles later, the PCR machine crashed, and was launched again. The bug is not expected to have a strong impact on the results.

On the late afternoon, a miniprep was made of pSB1C3 (S27), from the strains launched in the morning.

Camille & Danyel

Mathias and Laura already did the ligation between the T7 polymerase gene and its double terminator therefore we used their miniprep to do our QuickChange mutagenesis and on the same day launched an overnigth culture of the corresponding strain to keep the stock on.

The QuickChange mutagenesis mix was of :

  • 10µL of HF Buffer
  • 5µL of dNTP
  • 2µL oligo forward (initial solution concentration = 1pmol/µL)
  • 2µL oligo reverse (initial solution concentration = 1pmol/µL)
  • 5µL of the miniprep ([DNA]=55.9ng/µL)
  • 25.5µL of water

We digested the miniprep from yesterday of our strain containing PSB1C3+RFP to be able to use it for the ligation.
Then we run a gel and extracted the band of 2kb of PSB1C3, we then extracted it with a gel extraction kit the resulting [DNA]=9.4ng/µL.


We then did a ligation with a ratio 1 vector for 6 inserts and transformed it in competent E.coli, Axel asked us to do also .



Retrieved from "http://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/07/26/"