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Team:Paris Saclay/Notebook/July/31
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===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006'''==== | ===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006'''==== | ||
| - | ===='''1 -Digestion of | + | ===='''1 -Digestion of pSB1C3 by EcoRI/PstI'''==== |
Abdou | Abdou | ||
Used quantities : | Used quantities : | ||
| - | * | + | * pSB1C3 : 16µL |
* Buffer FD : 2µL | * Buffer FD : 2µL | ||
* EcoRI FD : 1µL | * EcoRI FD : 1µL | ||
| Line 21: | Line 21: | ||
We let our digestion 1h30 at 37°C. | We let our digestion 1h30 at 37°C. | ||
| - | ===='''2 - | + | ===='''2 - Inactivation of EcoRI/PstI used for the digestion of pSB1C3'''==== |
Abdou | Abdou | ||
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]] | Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]] | ||
| - | + | ||
| - | ===='''3 -Ligation of NarK, NarG, NirB in | + | ===='''3 -Ligation of NarK, NarG, NirB in pSB1C3'''==== |
XiaoJing | XiaoJing | ||
| Line 34: | Line 34: | ||
* Buffer : 2µL | * Buffer : 2µL | ||
* NarK, NarG, NirB : 1µL | * NarK, NarG, NirB : 1µL | ||
| - | * | + | * pSB1C3 : 2µL |
* Ligase : 1µL | * Ligase : 1µL | ||
* H2O : 14µL | * H2O : 14µL | ||
| - | ===='''4 -Electrophoresis to check the ligation of NarK, NarG, NirB in | + | ===='''4 -Electrophoresis to check the ligation of NarK, NarG, NirB in pSB1C3'''==== |
XiaoJing | XiaoJing | ||
{| | {| | ||
| - | | style="width:350px;border:1px solid black;" |[[]] | + | | style="width:350px;border:1px solid black;" |[[File:Psgel23107.jpg]] |
| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
* Well 1 : 6µL of DNA Ladder | * Well 1 : 6µL of DNA Ladder | ||
| Line 56: | Line 56: | ||
Expected sizes : | Expected sizes : | ||
| - | * | + | * Nar K : 200bp |
| - | * Nar G : | + | * Nar G : 200bp |
| - | * Nir B : | + | * Nir B : 200bp |
{| | {| | ||
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Expected sizes : | Expected sizes : | ||
* RBS-LacZ : 3093 bp | * RBS-LacZ : 3093 bp | ||
| - | * | + | * pSB1A2 : 2079 bp |
{| | {| | ||
Latest revision as of 01:00, 5 October 2013
Notebook : July 31
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006
1 -Digestion of pSB1C3 by EcoRI/PstI
Abdou
Used quantities :
- pSB1C3 : 16µL
- Buffer FD : 2µL
- EcoRI FD : 1µL
- PstI FD : 1µL
We let our digestion 1h30 at 37°C.
2 - Inactivation of EcoRI/PstI used for the digestion of pSB1C3
Abdou
Protocol : Ethanol precipitation
3 -Ligation of NarK, NarG, NirB in pSB1C3
XiaoJing
Used quantities :
- Buffer : 2µL
- NarK, NarG, NirB : 1µL
- pSB1C3 : 2µL
- Ligase : 1µL
- H2O : 14µL
4 -Electrophoresis to check the ligation of NarK, NarG, NirB in pSB1C3
XiaoJing
|
|
Expected sizes :
- Nar K : 200bp
- Nar G : 200bp
- Nir B : 200bp
|
We obtain fragments at the right size. We will transform them in DH5α. |
5 -Transformation of BBa_K1155004, BBa_K1155005, BBa_K1155006
Anaïs
Protocol : Bacterial transformation
Objective : obtaining BBa_K1155007
1 - Digestion of BBa_I732017 by EcoRI/SpeI
XiaoJing
Used quantities :
- BBa_I732017 : 41µL
- Buffer FD : 5µL
- EcoRI : 2µL
- SpeI : 2µL
We let the digestion at 1h30 at 37°C.
2 -Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI
XiaoJing
|
|
Expected sizes :
- RBS-LacZ : 3093 bp
- pSB1A2 : 2079 bp
|
We obtain fragments at the right size. We will purify it. |
3 - Gel purification of digestion of RBS-LacZ
Xavier
Protocol : Gel purification
|
We lost our fragments. We will do the digestion again. |
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