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

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June 8th, 2010

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

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).

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