User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/08

From OpenWetWare
Jump to: navigation, search
Igem-logo-150px.png iGEM UNAM-Genomics-Mexico Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

June 8th, 2010

1. Run gel to verify PCR for Blue Promoter and OGR from June 7th.

8June2010 PCR for Blue Promoter & OGR SUCCESSFUL.JPG

Lanes: 1,5) Ladder; 2) BP-1; 3) BP-2; 4) OGR.

  • PCR for Blue Promoter was finally successful!!! I’ll try the ligation with this PCR product and also with the designed primer (See “Minimum Blue Promoter.fasta” in Dropbox).
  • For the ligation with the PCR product I have to purify it and make a restiction assay with EcoRI and SpeI. After that I’ll have to make a ligation assay into a vector or a reporter (not our GFP reporter BBa_K145015 because it doesn´t have RBS yet).

2. We want to do an assay to measure fluorescence. Paz already has some strains with fluorescence proteins, these strains are:

  • B: BBa_K137018 GFP induced with acylhomoserinelactone (AHL).
  • D: BBa_K137019 GFP constitutively expressed, AHL shuts down GFP expression.
  • I: BBa_J23106 RFP under constitutive promoter.
  • H: eCFP without LVA tag. Under LacI promoter, induced with IPTG.
  • All these strains have to be re-cultured in solid LB medium with the respective antibiotic (all strains are resistant to Amp).
  • I have to look for a YFP reporter because we don’t have this fluorescence protein.
  • I will re-culture Paz’s strains when I have the transformation with YFP.

3. I made the PCR product purification using the High Pure PCR Product Purification Kit. I added 100ul of Elution buffer and tubes are marked: “Pure PCR BP1”; “Pure PCR BP2”; “Pure PCR OGR” and are also labeled with the date “8.JUN.10”.

4. Double restriction with EcoRI and SpeI to purified PCR product.

Double restriction methods:
  • DNA -> 5ul
  • Buffer 4 -> 2ul (10% of total volume)
  • BSA (required by SpeI) -> 1ul
  • EcoRI -> 1ul
  • SpeI -> 1ul
  • HPLC -> 10ul (to complete total volume of 20ul)
  • Incubate at 37ºC overnight

5. Meeting with Chris Wood

  • He doesn’t know about protocols to grow Vibrio Fischeri for luminiscence assay.
  • He recommended to use Promega kits for bioluminiscence assay, we have to check in detail these kits (Bright Glo, One Glo and Steady Glo).