Team:Grenoble/Biology/Notebook/June/week 28

From 2012.igem.org

(Difference between revisions)
Line 36: Line 36:
</ul>
</ul>
<br/>
<br/>
-
<h2>Tuesday, July A0<span class="exposant">th</span>:</h2>
+
<h2>Tuesday, July 10<span class="exposant">th</span>:</h2>
For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.<br/>
For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.<br/>
We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:

Revision as of 14:31, 6 August 2012

iGEM Grenoble 2012

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

Week 28: July 09th to July 15th

Goal of the week

we wanted to recover and amplify the biobricks involved in our genetic networks:
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • pLAC (100bp)
  • fha (80bp)
  • eCFP (800bp)
  • pLAC_RBS (120bp)
  • RsmA (200bp)
  • rsmY (170bp)
  • pSB1A3 (2400bp)
  • pSB4K5 (2400bp)
  • pSB3C5 (2400bp)
We also planned to realise the gibson assemblies for the first constructions.

Monday, July 09th:

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

Tuesday, July 10th:

For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.
We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
  • fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.
  • pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.
  • RBS_Cya from BW25113 WT precultured cells.
We did the PCR both with and without DMSO (Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO).

To separate the PCR products, we prepared two gels:
  • a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)
  • a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)

Migration conditions = 50V during 1h15.
We used EtBr to reveal the DNA fragments.

Retrieved from "http://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_28"