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Team:Paris Saclay/Notebook/July/26
From 2013.igem.org
(Difference between revisions)
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===='''Objective : obtaining Bba_K1155003'''==== | ===='''Objective : obtaining Bba_K1155003'''==== | ||
| - | ===='''1 - Ligation of RBS-AmilCP and Term-PSB1C3'''==== | + | ===='''1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : RBS-AmilCP '''==== |
| + | |||
| + | Abdou | ||
| + | |||
| + | Protocol : [[Team:Paris_Saclay/Protocols/Ethanol precipitation|Ethanol precipitation]] | ||
| + | |||
| + | We used 90µL of DNA. | ||
| + | |||
| + | ===='''2 - Ligation of RBS-AmilCP and Term-PSB1C3'''==== | ||
Xavier | Xavier | ||
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* RBS-AmilCP : 2µL | * RBS-AmilCP : 2µL | ||
| - | * | + | * Term in PSB1C3 : 2.5µL |
* Buffer : 1µL | * Buffer : 1µL | ||
* Ligase : 1µL | * Ligase : 1µL | ||
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| style="width:350px;border:1px solid black;" |[[]] | | style="width:350px;border:1px solid black;" |[[]] | ||
| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
| - | * Well 1 : 2µL of NirB+ | + | * Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye |
| - | * Well 2 : 2µL of NirB+1µL of 6X loading dye | + | * Well 2 : 2µL of NirB+3µL H2O+1µL of 6X loading dye |
| - | * Well 3 : 5µL of NarK+1µL of 6X loading dye | + | * Well 3 : 5µL of NarK+3µL H2O+1µL of 6X loading dye |
| - | * Well 4 : 5µL of NarK+1µL of 6X loading dye | + | * Well 4 : 5µL of NarK+3µL H2O+1µL of 6X loading dye |
* Well 5 : 6µL DNA Ladder | * Well 5 : 6µL DNA Ladder | ||
| - | * Well 6 : 5µL of NarG+1µL of 6X loading dye | + | * Well 6 : 5µL of NarG+3µL H2O+1µL of 6X loading dye |
| - | * Well 7 : 5µL of NarG+1µL of 6X loading dye | + | * Well 7 : 5µL of NarG+3µL H2O+1µL of 6X loading dye |
* Gel : 1.2% | * Gel : 1.2% | ||
|} | |} | ||
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Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]] | Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]] | ||
| - | REPRIS dans 20µL !!!!!!!! | + | REPRIS dans 20µL !!!!!!!! |
| + | |||
| + | ===='''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. | ||
| + | |||
{{Team:Paris_Saclay/incl_fin}} | {{Team:Paris_Saclay/incl_fin}} | ||
Revision as of 15:46, 15 September 2013
Notebook : July 26
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining Bba_K1155003
1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : RBS-AmilCP
Abdou
Protocol : Ethanol precipitation
We used 90µL of DNA.
2 - 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 : ...
|
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 : cf PCR précédente !!!!!!!!!!!!! ... ADN non dilué
PCR program : cf PCR précédente !!!!!!!!! SCHEMA !!!!!!!!!!!!!!
3 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou, Xavier
| [[]] |
|
Expected sizes :
- NarK, NarG, NirB : ...
|
We obtain fragments at the right size. We can purify it. |
4 - Gel purification of PCR products : NarK, NarG, NirB
Xavier
Protocol : Gel purification
REPRIS dans 20µL !!!!!!!!
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.
