User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/07

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June 7th, 2010

1. Design primer to add RBS to GFP reporter BBa_K145015.

  • This reporter starts with “atg” and its length is 759 bp.
  • There’s already a primer for this purpose, I’ll check it and correct it if necessary.
  • The existing primer anneal 20 bp from position 1 to 20 in the reporter sequence. It has a Tm of 48.6ºC, a GC content of 40%, 53 bp length and ends with “TT”. I’ll add some base pairs to the 3’ end to increase the GC content and make it end with a G or C.
  • I added 9 bp to the primer, it finally anneals with 29 bp in the reporter. I also reduced the Preffix region of the primer to leave it only with the XbaI restriction site.

2. Wet lab meeting:

  • Last week we decided to change the way to extract the Blue Promoter, we were using PCR, now we decided to synthezise an oligo containing the preffix to prime plasmid 30, the minimum blue promoter (50bp), and a restriction site for SpeI.
  • Check in a gel the DNA extraction made by Miguel, it was for genomic DNA from E. coli K12 and Vibrio Fischeri MJ11.
  • Luminiscence assay with Vibrio Fischeri, using a luminometer and the cameras provided by Alberto Soria. IT WILL BE DONE BY ME. I have to check how this assay is made (Protocols to grow the bacteria, incubation temperature, etc.).
  • Ask Claudia to talk to Alberto Soria and ask him for the cameras to make the Vibrio Fischeri luminiscence assay.

3. Run gel to verify the DNA extraction from E. coli K12 and Vibrio Fischeri MJ11 strains made by Miguel.

  • Agarose gel at 0.8%, run for 50 min at 90V.
7Jun2010 K12 & VF MJ11 DNA extraction by Miguel.JPG

Lanes: 1,6) Ladder; 2) E. coli K12-1; 3) E. coli K12-2; 4) Vibrio Fischeri MJ11-1; 5) Vibrio Fischeri MJ11-2.

4. Repeat PCR for Blue Promoter (and OGR as control) with the new genomic DNA extracted by Miguel.

  • Reactives needed for two reactions to amplify Blue Promoter:
Taq Polymerase -> 1
Taq Reaction Buffer -> 5
MgCl 50mM (can be used up to 3ul) -> 2.5
dNTP’s 0.4ug/ul -> 2.5
Primer Forward (can be used up to 3ul) -> 2.5
Primer Reverse (can be used up to 3ul) -> 2.5
HPLC -> 32
DNA -> 2
Total volume -> 50