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Team:Paris Saclay/Notebook/July/26
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===='''Objective : obtaining BBa_K1155003'''==== | ===='''Objective : obtaining BBa_K1155003'''==== | ||
| - | ===='''1 - | + | ===='''1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP '''==== |
Abdou | Abdou | ||
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{| | {| | ||
| - | | style="width:350px;border:1px solid black;" |[[]] | + | | style="width:350px;border:1px solid black;" |[[File:Psgel2607.jpg]] |
| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
* Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye | * Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye | ||
| Line 31: | Line 31: | ||
Expected sizes : | Expected sizes : | ||
| - | * RBS-AmilCP : | + | * RBS-AmilCP : 3500bp |
{| | {| | ||
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|} | |} | ||
| - | + | ===='''3 - Ligation of RBS-AmilCP and Term-pSB1C3'''==== | |
| - | ===='''3 - Ligation of RBS-AmilCP and Term- | + | |
Xavier | Xavier | ||
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* RBS-AmilCP : 2µL | * RBS-AmilCP : 2µL | ||
| - | * Term in | + | * Term in pSB1C3 : 2.5µL |
* Buffer : 1µL | * Buffer : 1µL | ||
* Ligase : 1µL | * Ligase : 1µL | ||
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| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
* Well 1 : 5µL of NirB+1µL of 6X loading dye | * Well 1 : 5µL of NirB+1µL of 6X loading dye | ||
| - | * Well 2 : 5µL of NirB+1µL of 6X loading dye | + | * Well 2 : 5µL of NirB+1µL of 6X loading dye |
* Well 3 : 5µL of NarG+1µL of 6X loading dye | * Well 3 : 5µL of NarG+1µL of 6X loading dye | ||
* Well 4 : 5µL of NarG+1µL of 6X loading dye | * Well 4 : 5µL of NarG+1µL of 6X loading dye | ||
* Well 5 : 6µL DNA Ladder | * Well 5 : 6µL DNA Ladder | ||
| - | * Well 6 : 5µL of NarK+1µL of 6X loading dye | + | * Well 6 : 5µL of NarK+1µL of 6X loading dye |
* Well 7 : 5µL of NarK+1µL of 6X loading dye | * Well 7 : 5µL of NarK+1µL of 6X loading dye | ||
* Well 8 : 5µL of NarK+1µL of 6X loading dye | * Well 8 : 5µL of NarK+1µL of 6X loading dye | ||
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Expected sizes : | Expected sizes : | ||
| - | * NarK, NarG, NirB : | + | * NarK, NarG, NirB : 200bp |
{| | {| | ||
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** Buffer phusion : 10µL | ** Buffer phusion : 10µL | ||
** dNTP : 1µL | ** dNTP : 1µL | ||
| - | ** Oligo | + | ** Oligo 47 : 2µL |
| - | ** Oligo | + | ** Oligo 48 : 2µL |
** Concentrated DNA : 2µL | ** Concentrated DNA : 2µL | ||
** Phusion : 0.25µL | ** Phusion : 0.25µL | ||
| Line 96: | Line 95: | ||
** Buffer phusion : 10µL | ** Buffer phusion : 10µL | ||
** dNTP : 1µL | ** dNTP : 1µL | ||
| - | ** Oligo | + | ** Oligo 49 : 2µL |
| - | ** Oligo | + | ** Oligo 50 : 2µL |
** Concentrated DNA : 2µL | ** Concentrated DNA : 2µL | ||
** Phusion : 0.25µL | ** Phusion : 0.25µL | ||
| Line 104: | Line 103: | ||
** Buffer phusion : 10µL | ** Buffer phusion : 10µL | ||
** dNTP : 1µL | ** dNTP : 1µL | ||
| - | ** Oligo | + | ** Oligo 45 : 2µL |
| - | ** Oligo | + | ** Oligo 46 : 2µL |
** Concentrated DNA : 2µL | ** Concentrated DNA : 2µL | ||
** Phusion : 0.25µL | ** Phusion : 0.25µL | ||
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Expected sizes : | Expected sizes : | ||
| - | * NarK, NarG, NirB : | + | * NarK, NarG, NirB : 200bp |
{| | {| | ||
Latest revision as of 00:29, 5 October 2013
Notebook : July 26
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155003
1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP
Abdou
Protocol : Ethanol precipitation
We used 90µL of DNA. At the end, we mix our DNA with 20µL of water.
2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP
Abdou
|
|
Expected sizes :
- RBS-AmilCP : 3500bp
|
We obtain fragments at the right size. We will ligate RBS-AmilCP with Term. |
3 - Ligation of RBS-AmilCP and Term-pSB1C3
Xavier
Used quantities :
- RBS-AmilCP : 2µL
- Term in pSB1C3 : 2.5µL
- Buffer : 1µL
- Ligase : 1µL
- H2O : 3.5µL
Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou
|
|
Expected sizes :
- NarK, NarG, NirB : 200bp
|
We lost all our PCR products. We will do PCR again using more concentrated DNA and oligos. |
2 - PCR of NarK, NarG, NirB
Abdou, Xavier
Used quantities :
- NarK :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo 47 : 2µL
- Oligo 48 : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
- NarG :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo 49 : 2µL
- Oligo 50 : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
- NirB :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo 45 : 2µL
- Oligo 46 : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
PCR Program :
3 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou, Xavier
|
|
Expected sizes :
- NarK, NarG, NirB : 200bp
|
We obtain fragments at the right size. We can purify it. |
4 - Gel purification of PCR products : NarK, NarG, NirB
Xavier
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
At the end we mix our DNA in 20µL of H2O.
5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI
Abdou
Quantities used :
- NarK, NarG, NirB : 10µL
- Buffer FD : 3µL
- EcoRI FD : 1.5µL
- PstI FD : 1.5µL
- H2O : 14µL
We let the digestion at 37°C during 1h30 and then at -20°C.
B - PCB sensor system
Objective : obtaining BBa_K1155002
1 - Tranformation of BBa_K1155002 in DH5α
Abdou, Xavier
Protocol : Bacterial tranformation
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