Team:Grenoble/Biology/Notebook/August/week 34

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iGEM Grenoble 2012

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August

Week 31 • Week 32 • Week 33 • Week 34 • Week 35

Week 34: August 20th to 26th

Goal of the week:

Assembly of:
  • pAra/Bad_RBS_GFP and RBS_Cya.
  • pompC and mcherry
  • Tap and EnvZ

Monday, August 20th:

We did some verification PCRs on miniprep with HF Phusion enzyme (protocol) in order to check the Gibson Assembly products (12/09/09>.
Annealing temperature = 55°C.

To separate (protocol) the PCR 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.

It showed no significant results (data not shown).

Tuesday, August 21th:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.

We transformed (new protocol) BW25113 WT competent cells (protocol) with pSB3C5.

Wednesday, August 22nd:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. The M9 medium used was complemented: 0.1% acetate.

Friday, August 24rd:

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pOmpC with EcoRI and SpeI during 10 minutes
  • pOmpC with EcoRI during 10 minute
  • pSB4K5 with EcoRI and SpeI during 10 minutes
  • pSB4K5 with EcoRI during 10 minutes

To separate (protocol) the digestion 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.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products and from the pOmpC and pSB4K5 digestion products.

We did some PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4C5 (mcherry), pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pSB4C5 with EcoRI and PstI during 10 minutes
  • pSB4K5 with EcoRI and PstI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes

To separate (protocol) the PCR and the digestion 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.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.

We realised a Gibson Assembly (protocol) to build:
  • pSB4K5 with Tap-EnvZ
  • pSB3C5 with pAra/Bad_RBS_GFP_RBS_Cya

We did some digestions (protocol) on pSB3C5, pSB4K5 and pSB1A3. The digestions were achieved with two restriction enzymes: EcoRI and PstI during 10 minutes.

To separate (protocol) the PCR and the digestion 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.

The digestion worked well, we realised a DNA extraction (protocol) from the pSB1A3 and pSB3C5 products.

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