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Team:Grenoble/Biology/Notebook/July/week 33
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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.
