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Team:Grenoble/Biology/Notebook/August/week 33
From 2012.igem.org
(Difference between revisions)
| Line 51: | Line 51: | ||
<section> | <section> | ||
<h2>Thursday, August 16<span class="exposant">th</span>:</h2> | <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/> | + | 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/> | <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: | 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> pOmpC with EcoRI and SpeI during 10 minutes </li> | <li> pOmpC with EcoRI and SpeI during 10 minutes </li> | ||
| - | <li> pOmpC with EcoRI</li> | + | <li> pOmpC with EcoRI during 10 minute</li> |
<li> pSB4K5 with EcoRI and SpeI during 10 minutes</li> | <li> pSB4K5 with EcoRI and SpeI during 10 minutes</li> | ||
<li> pSB4K5 with EcoRI during 10 minutes</li> | <li> pSB4K5 with EcoRI during 10 minutes</li> | ||
| Line 65: | Line 65: | ||
In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<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/> | <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/> | + | 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 and from the pOmpC and pSB4K5 digestion products.<br/> |
<br/> | <br/> | ||
| + | We did some PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB4C5 (mcherry), pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.<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> pSB4C5 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> | ||
| + | </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 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/> | ||
| + | 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 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 and pSB3C5 products.<br/> | ||
| + | </section> | ||
| + | <section> | ||
| + | <h2>Friday, August 17<span class="exposant">th</span>:</h2> | ||
Revision as of 23:09, 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 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.
