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Team:Grenoble/Biology/Notebook/July/week 33
From 2012.igem.org
(Difference between revisions)
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We transformed (new <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT | We transformed (new <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT | ||
competent cells (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) with pSB3C5.<br/> | competent cells (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) with pSB3C5.<br/> | ||
| - | <br/> | + | </section> |
| + | |||
| + | <section> | ||
| + | <h2> Wednesday, August 15<span class="exposant">th</span>:</h2> | ||
| + | We did an experiment (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test">protocol</a>) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. The M9 medium used was complemented: 0.1% acetate. | ||
| + | </section> | ||
| + | |||
| + | <section> | ||
| + | <h2>Thursday, August 16<span class="exposant">th</span>:</h2> | ||
| + | We did some colony PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB1A3 (araC), pSB3C5 (araC), pSB3C5 (pAra/Bad_RBS_GFP_RBS_Cya) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya)<br/> | ||
| + | <br/> | ||
| + | Then, we did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes: | ||
| + | <ul> | ||
| + | <li> pOmpC with EcoRI and SpeI during 10 minutes </li> | ||
| + | <li> pOmpC with EcoRI</li> | ||
| + | <li> pSB4K5 with EcoRI and SpeI during 10 minutes</li> | ||
| + | <li> pSB4K5 with EcoRI during 10 minutes</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR and the digestion products, we prepared a 1.8% TAE agarose gel.<br/> | ||
| + | Migration conditions = 100V during 30 min.<br/> | ||
| + | In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/> | ||
| + | <br/> | ||
| + | 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 pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products.<br/> | ||
| + | <br/> | ||
| + | |||
| + | |||
</div> | </div> | ||
Revision as of 22:52, 26 September 2012
August
Week 31 • Week 32 • Week 33 • Week 34 • Week 35Week 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
- 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.
