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Team:Paris Saclay/Notebook/July/3
From 2013.igem.org
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Tranformation of 07/02/13 works. We will do a Colony PCR of colonies. | Tranformation of 07/02/13 works. We will do a Colony PCR of colonies. | ||
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<center>[[File:PSprimer07.jpg|right|300px]]</center> | <center>[[File:PSprimer07.jpg|right|300px]]</center> | ||
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| + | Colonies count : | ||
* Standard concentration : | * Standard concentration : | ||
Revision as of 14:32, 22 September 2013
| colonies | Normal concentration | High concentration |
| control | ||
| Fnr in plasmid PSB1C3 |
We picked up 4 colonies for further test (2 include FNR+plasmid in PSB1C3 and 2 from the control)
Primer and PCR
VF2, VR, Pfnr_up, Pfnr_down are 4 primes that we used for plasmid restriction. The primers, if the promoter fnr entre the plasmid successfully will amplify 3 sub-pieces with specific size. They are VF/VR, VF/Pfnr_down, Pfnr_up/VR.
So we had prepared 4(colonies)*3(amplification) = 12 PCR tubes.
- Dream Taq(5µg/µl):2µl
- Buffer (Dream Taq) 10X:10µl
- dNTP:2µl
- Primer (F/R;F/fnr_R;fnr_F/R):2µl+2µl
- H2O:82µl
- Total:100µl (volume for 4 tubes, so 25µl for each)
PCR programe
The PCR products was put on a gel of agarose 1.5% with BET (1.5%), migrated during 30 min at 100V. the picture analysis was be delayed to the next day.
Culture confirmation
We performed another colonies seeding. 2 colonies selected from each Petri dish were seeded in liquid medium with their corresponding antibiotic at 37°C under stirring during 1 night.
Contents |
Notebook : July 1
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining Bba_K1155000
1 - Colony PCR of Bba_K1155000, Bba_I732017, Bba_K592009
Abdou, Sheng, Zhou
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Tranformation of 07/02/13 works. We will do a Colony PCR of colonies. |
Colonies count :
- Standard concentration :
- Bba_K1155000 : 0
- High concentration :
- Bba_K1155000 : 2
Primer and PCR :
VF2, VR, Pfnr_up, Pfnr_down are four oligos that we used for plasmid amplification. We used tree combinaisons VF/VR, VF/Pfnr_Down, Pfnr_Up/VR. If the promoter Pfnr insert PSB1C3 plasmid successfully, tree fragments with specific size will be amplified.
COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.
Used quantities :
- DNA : 2µL
- Mix : (it was divided in tubes for 4 different colonies for each oligo combinaison with 23µL of mix in each tube)
- VF or Pfnr_Up : 6µL
- VR or Pfnr_Down or VR : 6µL
- dNTP : 6µL
- Buffer Dream Taq : 30µL
- Dream Taq : 6µL
- H2O : 246µL
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