Team:Grenoble/Biology/Notebook/July/week 29

From 2012.igem.org

Revision as of 15:31, 6 August 2012 by Adeline millet (Talk | contribs)

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project
week 27 week 28 week 29 week 30

Week 29: July 16th to July 22nd

Goal of the week

test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks :
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • fha1 (80bp)
  • eCFP (800bp)
  • pSB4K5 (2400bp)

Monday, July 16th:

Precultured cells are prepared:
  • Strains = BW25113 WT and BW25113 cya- pAra/Bad
  • Conditions = Conditions = LB liquid medium, 37°C, 200rpm, overnight

Tuesday, July 17th:

Using iGEM 2012 biobricks and the Gibson Assembly product we transformed (protocol) BW25113 WT cells. We obtained four transformed strains with:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • 120713PP_GA_001 (rsmY)
  • 120713PP_GA_002 (RsmA)

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains, on which we wanted to recover pSB4K5 and the plasmid with pAra/Bad_RBS_GFP:
  • transformed strain with pSB4K5
  • BW25113 cya- pAra/Bad

We did PCRs with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB4K5, and from colony to amplify pLAC (fha), pLAC (rsmY), pLAC_RBS, fha and pSB4K5. We did the PCR both with and without DMSO.

To separate the PCR products and the miniprep products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

Retrieved from "http://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_29"