Latest revision as of 03:26, 27 September 2012

iGEM Grenoble 2012

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August

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

Week 33: August 13th to 19th

Goal of the week:

Assembly of:

pAra/Bad_RBS_GFP and RBS_Cya.
pompC and mcherry
Tap and EnvZ

Monday, August 13th:

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 14th:

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 15th:

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.

Thursday, August 16th:

We did some colony PCRs with HF Phusion enzyme (protocol) in order to amplify pSB1A3 (araC), pSB3C5 (araC), pSB3C5 (pAra/Bad_RBS_GFP_RBS_Cya) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya).

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 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 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 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.

@@ Line 83: / Line 83: @@
 The PCR and the digestion worked well, we realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.<br/>
 <br/>
-We realised a Gibson Assembly (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/GA">protocol</a>) to build: <ul><li>
- pSB4K5 with Tap-EnvZ</li>
-<li>pSB3C5 with pAra/Bad_RBS_GFP_RBS_Cya</li></ul><br/>
 We did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) on pSB3C5, pSB4K5 and pSB1A3. The digestions were achieved with two restriction enzymes: EcoRI and PstI during 10 minutes.<br/>
 <br/>

Team:Grenoble/Biology/Notebook/August/week 33

From 2012.igem.org