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Notebook : July 3

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155000

**1 - Colony PCR of BBa_K1155000 to check good insertion of Pndh* in pSB1C3**

Abdou, Sheng, Zhou

Tranformation of 07/02/13 works. We will do a PCR colony.

Colonies count for BBa_K1155000 :

Standard concentration :
- BBa_K1155000 : 0 colony

High concentration :
- BBa_K1155000 : 2 colonies

Primers 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 Pndh* insert pSB1C3 plasmid successfully, tree fragments with specific size will be amplified.

Protocol

We resuspend each colony with 20µL of H2O.

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 : 6µL
- dNTP 10mM : 6µL
- Buffer Dream Taq : 30µL
- Dream Taq : 6µL
- H2O : 246µL

2 - Culture of BBa_K1155000

Sheng

We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_K1155000. We also did liquid culture of each one. We incubate culture at 37°C.

Objective : obtaining BBa_K1155003, BBa_K1155007

1 - Culture of BBa_I732017, BBa_K592009

Sheng

Tranformation of 07/02/13 works. We will do new cultures.

We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_I732017, BBa_K592009. We also did liquid culture of each one. We incubate culture at 37°C.

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 {{Team:Paris_Saclay/incl_debut_generique}}
 ='''Notebook : July 3'''=
+=='''Lab work'''==
-=='''Summary:'''==
+==='''A - Aerobic/Anaerobic regulation system'''===
-FNR regulator system:
-*Continued what we started yesterday. Observed the Petri dish, selected the colonies. 4 colonies in total, they were 2 include FNR+plasmid in PSB1C3 and 2 from the control.
+===='''Objective : obtaining BBa_K1155000'''====
-*Used 4 primers: VF2, VR, Pfnr_up, Pfnr_down for the verification test. They were designed to cut 4 special sites for creating 3 different regions on plasmid chain: VF2/VR, VF2/PFNR_down, PFNR_up/VR. After the amplification, those PCR products had been put on electrophoresis gel for the verification.
-=='''Lab work'''==
+===='''1 - Colony PCR of BBa_K1155000 to check good insertion of Pndh* in pSB1C3'''====
-*A.aero/anaerobic regulation system
+Abdou, Sheng, Zhou
-:*2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
-:*BioBrick RBS+amilCP+terminator in plasmid PSB1C3
-<u>Observation</u>
+{|
+| style="border:1px solid black;padding:5px;background-color:#DE;" |
+Tranformation of 07/02/13 works. We will do a PCR colony.
+|}
-<p>After the incubation overnight, we observed the Petri dishes. </p>
+Colonies count for BBa_K1155000 :
-{| border="1" align="center"
+* Standard concentration :
-|-
+** BBa_K1155000 : 0 colony
-|colonies|Normal concentration|High concentration
-|-
-|control|11|60
-|-
-|Fnr in plasmid PSB1C3|0|2
-|}
-colonies	Normal concentration	High concentration
-control	11	60
- Fnr in plasmid PSB1C3	0	2
-We picked up 4 colonies for further test (2 include FNR+plasmid in PSB1C3 and 2 from the control)
+* High concentration :
-PCR et Primer
+** BBa_K1155000 : 2 colonies
-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.
+[[File:Ps0307jour.jpg|300px]]
-We had prepared 4(colonies)*3(amplification) = 12 PCR tubes.
+<br>
+<center>[[File:PSprimer07.jpg|right|250px]]</center>
+<br>
+'''[https://2013.igem.org/Team:Paris_Saclay/Primers Primers] and PCR :'''
+<p>'''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 Pndh* insert pSB1C3 plasmid successfully, tree fragments with specific size will be amplified.''' </p>
+'''
+Protocol'''
-Dream Taq(5µg/µl)……………………..2µl
+We resuspend each colony with 20µL of H2O.
-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 was programed for 95°C for 3mins and 30 cycles of 94°C 30s + 58°C 30s + 72°C 30s.
+Used quantities :
-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.
+* DNA : 2µL
-Culture confirmation
+* Mix : (it was divided in tubes for 4 different colonies for each oligo combinaison with 23µL of mix in each tube)
-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.
+** VF or Pfnr_Up : 6µL
+** VR or Pfnr_Down : 6µL
+** dNTP 10mM : 6µL
+** Buffer Dream Taq : 30µL
+** Dream Taq : 6µL
+** H2O : 246µL
+[[File:PSPCR0307.jpg|400px]]
+===='''2 - Culture of BBa_K1155000 '''====
+Sheng
+We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_K1155000. We also did liquid culture of each one.
+We incubate culture at 37°C.
+===='''Objective : obtaining BBa_K1155003, BBa_K1155007'''====
+===='''1 - Culture of BBa_I732017, BBa_K592009 '''====
+Sheng
+{|
+| style="border:1px solid black;padding:5px;background-color:#DE;" |
+Tranformation of 07/02/13 works. We will do new cultures.
+|}
+We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_I732017, BBa_K592009. We also did liquid culture of each one.
+We incubate culture at 37°C.
-<br>
 {| border="1" align="center"
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