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Team:Grenoble/Biology/Notebook/August/week 33
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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: | 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> | <ul> | ||
| - | <li> pSB4C5 with EcoRI and PstI during 10 minutes </li> | + | <li> pSB4C5 with EcoRI and PstI during 10 minutes</li> |
| - | <li> pSB4K5 with EcoRI and PstI during 10 minutes </li> | + | <li> pSB4K5 with EcoRI and PstI during 10 minutes</li> |
<li> mcherry with PstI and XbaI during 10 minutes</li> | <li> mcherry with PstI and XbaI during 10 minutes</li> | ||
<li> mcherry with PstI and XbaI during 10 minutes</li> | <li> mcherry with PstI and XbaI during 10 minutes</li> | ||
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Revision as of 00:06, 27 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 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 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.
