Team:Paris Saclay/Notebook/July/4

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : obtaining Bba_K1155000'''====
+
===='''Objective : obtaining BBa_K1155000'''====
-
===='''1 - Estimation of Pfnr Colony PCR size fragments'''====
+
===='''1 - Estimation of Pndh* Colony PCR size fragments'''====
Abdou, Anaïs, Sheng
Abdou, Anaïs, Sheng
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Estimated size :  
Estimated size :  
-
* VF/VR primer -> Plasmid without Pfnr size : 277bp
+
* VF/VR primer -> Plasmid without Pndh* size : 277bp
-
* VF/Pfnr_down -> Plasmid with Pfnr size : 276bp
+
* VF/Pfnr_down -> Plasmid with Pndh* size : 276bp
-
* Pfnr_Up/VR -> Plasmid with Pfnr size : 311bp
+
* Pfnr_Up/VR -> Plasmid with Pndh* size : 311bp
-
===='''1 - Electrophoresis of Colony PCR products'''====
+
===='''2 - Electrophoresis of Colony PCR products'''====
Zhou
Zhou
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| style="width:350px;border:1px solid black;vertical-align:top;" |
| 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 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 5 to 6 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
* Well 7 : 6µL of DNA Ladder
* Well 7 : 6µL of DNA Ladder
-
* Well 8 to 9 : 5µL of PCR product withVF/Pfnr_Down primers+1µl of 6X loading dye
+
* 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 10 to 13 : 5µL of PCR products with Pfnr_Up/VR primers+1µL of 6X loading dye
* Well 14 : 6µL of DNA Ladder
* Well 14 : 6µL of DNA Ladder
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| 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 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.
-
WE OBTAIN OUR FIRST BIOBRICK ! We call it Bba_K1155000.
+
|}
|}
-
===='''2 - Stock of Bba_K1155000'''====
+
===='''3 - Stock of BBa_K1155000'''====
Zhou
Zhou
Used quantities :  
Used quantities :  
-
* Bba_K1155000 confirmed : 1 mL
+
* BBa_K1155000 confirmed : 1 mL
-
* Glycérol : 500µL glycerol.  
+
* Glycerol : 500µL glycerol.  
We stocked them at -20°C.
We stocked them at -20°C.
-
===='''3 - Extraction of Bba_K1155000 from DH5α'''====
+
===='''4 - Extraction of BBa_K1155000 from DH5α'''====
Anaïs, Sheng
Anaïs, Sheng
-
Protocol : [[Team:Paris_Saclay/Protocols/Hight copy plamid extraction|Hight copy plamid extraction]]
+
Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
-
===='''3 - Digestion of Bba_K1155000 by NotI, MluI, HpaI'''====
+
===='''5 - Digestion of BBa_K1155000 by NotI, MluI, HpaI to check good insertion of Pndh* in pSB1C3'''====
Abdou
Abdou
Used quantities :  
Used quantities :  
-
* Bba_K1155000 : 2µL
+
* BBa_K1155000 : 2µL
* Bufer oranger : 2µL
* Bufer oranger : 2µL
* NotI or MluI or HpaI : 0.5µL
* NotI or MluI or HpaI : 0.5µL
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We let the digestion 1h30 at 37°C.
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.
 +
{| border="1" align="center"
{| border="1" align="center"

Latest revision as of 16:35, 4 October 2013

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Contents

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

Psgel0407.jpg
  • 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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