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Notebook : July 4

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155000

**1 - Estimation of Pndh* Colony PCR size fragments**

Abdou, Anaïs, Sheng

We used software gene manager to find the correct size of our fragments.

Estimated size :

VF/VR primer -> Plasmid without Pndh* size : 277bp
VF/Pfnr_down -> Plasmid with Pndh* size : 276bp
Pfnr_Up/VR -> Plasmid with Pndh* size : 311bp

2 - Electrophoresis of Colony PCR products

Zhou

Well 1 to 4 : 5µL of PCR products with VF/VR primers+1µL of 6X loading dye
Well 5 to 6 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
Well 7 : 6µL of DNA Ladder
Well 8 to 9 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
Well 10 to 13 : 5µL of PCR products with Pfnr_Up/VR primers+1µL of 6X loading dye
Well 14 : 6µL of DNA Ladder
Gel : 1.5%

We obtain fragment at the right size in wells : 3, 4 , 8 , 9 12 and 13. We will digest our biobrick to confirm that Pndh* insert pSB1C3 correctly.

3 - Stock of BBa_K1155000

Zhou

Used quantities :

BBa_K1155000 confirmed : 1 mL
Glycerol : 500µL glycerol.

We stocked them at -20°C.

4 - Extraction of BBa_K1155000 from DH5α

Anaïs, Sheng

Protocol : Hight copy plamid extraction

**5 - Digestion of BBa_K1155000 by NotI, MluI, HpaI to check good insertion of Pndh* in pSB1C3**

Abdou

Used quantities :

BBa_K1155000 : 2µL
Bufer oranger : 2µL
NotI or MluI or HpaI : 0.5µL
H2O : 15.5µL

We let the digestion 1h30 at 37°C.

6 - Culture of BBa_K1155000

Anaïs, Sheng

We made 2 cultures of bacterias transformed with BBa_K1155000 that show a fragments at the right size at electrophoresis.

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@@ Line 1: / Line 1: @@
 {{Team:Paris_Saclay/incl_debut_generique}}
 ='''Notebook : July 4'''=
+=='''Lab work'''==
-===''Summary: ''===
+==='''A - Aerobic/Anaerobic regulation system'''===
-<div>For regulator system:
-*estimated the size of segments for bands of electrophoresis by using Clone Manager.
-*Made interpretation for electrophoresis. The picture shows that the experimental result was coherent with our estimation. We constructed our first BrioBrick, BBa_K1155000 (fnr+plasmid PSB1C3).
-*stored 2 colonies who contain BioBrick BBa_K1155000
-*the plasmid DNA extraction was performed for BBa_K1155000.
-*The extract of plasmid DNA which contain BBa_K1155000 was digested by Not I, Mlu I, Hpa I, and were amplified by PCR.
-*Seeded the 4 additional broths (2 for amilCP, 2 for FNR) for plasmid extraction(mini prep).
-For PCBs sensor system:
-*Received the bacterial strain: pseudomonas KE707.</div>
-=='''Lab work'''==
+===='''Objective : obtaining BBa_K1155000'''====
-constructing
+===='''1 - Estimation of Pndh* Colony PCR size fragments'''====
+Abdou, Anaïs, Sheng
+We used software gene manager to find the correct size of our fragments.
+Estimated size :
+* VF/VR primer -> Plasmid without Pndh* size : 277bp
+* VF/Pfnr_down -> Plasmid with Pndh* size : 276bp
+* Pfnr_Up/VR -> Plasmid with Pndh* size : 311bp
+===='''2 - Electrophoresis of Colony PCR products'''====
+Zhou
+{|
+| style="width:350px;border:1px solid black;" |[[File:Psgel0407.jpg]]
+| style="width:350px;border:1px solid black;vertical-align:top;" |
+* Well 1 to 4 : 5µL of PCR products with VF/VR primers+1µL of 6X loading dye
+* Well 5 to 6 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
+* Well 7 : 6µL of DNA Ladder
+* Well 8 to 9 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
+* Well 10 to 13 : 5µL of PCR products with Pfnr_Up/VR primers+1µL of 6X loading dye
+* Well 14 : 6µL of DNA Ladder
+* Gel : 1.5%
+|}
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We obtain fragment at the right size in wells : 3, 4 , 8 , 9 12 and 13. We will digest our biobrick to confirm that Pndh* insert pSB1C3 correctly.
+|}
+===='''3 - Stock of BBa_K1155000'''====
+Zhou
+Used quantities :
+* BBa_K1155000 confirmed : 1 mL
+* Glycerol : 500µL glycerol.
+We stocked them at -20°C.
+===='''4 - Extraction of BBa_K1155000 from DH5α'''====
+Anaïs, Sheng
+Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
+===='''5 - Digestion of BBa_K1155000 by NotI, MluI, HpaI to check good insertion of Pndh* in pSB1C3'''====
+Abdou
+Used quantities :
+* BBa_K1155000 : 2µL
+* Bufer oranger : 2µL
+* NotI or MluI or HpaI : 0.5µL
+* H2O : 15.5µL
+We let the digestion 1h30 at 37°C.
+===='''6 - Culture of BBa_K1155000'''====
+Anaïs, Sheng
+We made 2 cultures of bacterias transformed with BBa_K1155000 that show a fragments at the right size at electrophoresis.
-<br>
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