Team:Paris Liliane Bettencourt/Notebook/2011/08/25/

From 2011.igem.org

Team IGEM Paris 2011

Contents

Cyrille

YFP cloning

6 colonies from the ligation plate were taken and miniprep overday. The tube where prepared for the sequencing.

As there where very few µL left in the miniprep, a tranformation was done again and plated.

YFP QCM

The PCR product was digested one hour using fast digest DpnI. A gel was runned.

Ladder - Non digested minus control - negative control - 5ng - 25 ng - 50ng

Then, the product was transformed. As I was not sure for one pipeting, two tubes where done for the 5ng plate. The result is plated and growth overnight.

There where no colonies on the control and several in tge 25 and 50 ng as expected from the gel

Axel

knock out of CodY gene of the strain php13-v10-6b

strain CodY::Spec is CodY- We have to extract genomic DNA of that strain using the following protocol (phenol/chloroform one): http://userpages.umbc.edu/~jwolf/m1.htm

strain PHP13-V10-6B is CodY+ Make competent cells of that strain using the starvation protocol (https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/07/27/)

Transformation of php13-v10-6b with extracted genomic DNA. We expect a recombination between CodY::Spec gene and CodY gene. Thus we should "knock out" php13-v10-6b. Results on plates where unconsistent.

Camille

I digested the minipreps from yesterday, approximatively 1µg. Vectors were here :

  • S27 : pSB1C3
  • S46 : pSB1C3 + tRNAamb + double terminator
  • S58 : pSB1C3 + RBS spoVG

After digestion, I ran a gel, the bands were good, so I did a gel extraction.

Strain [DNA] in ng/µL ratio
S27 18.1 3.29
S46 20 2.5
S58 6.9 3.14

For the ligation I used a vector mass of 100ng.
Then I transformed with a ratio of one vector for six inserts.





Retrieved from "http://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/25/"