Team:Paris Saclay/Notebook/July/4

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : July 4'''=
='''Notebook : July 4'''=
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=='''Lab work'''==
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===''Summary: ''===
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==='''A - Aerobic/Anaerobic regulation system'''===
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<div>For regulator system:
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*estimated the size of segments for bands of electrophoresis by using Clone Manager.
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*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).
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*stored 2 colonies who contain BioBrick BBa_K1155000
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*the plasmid DNA extraction was performed for BBa_K1155000.
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*The extract of plasmid DNA which contain BBa_K1155000 was digested by Not I, Mlu I, Hpa I, and were amplified by PCR.
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*Seeded the 4 additional broths (2 for amilCP, 2 for FNR) for plasmid extraction(mini prep).
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For PCBs sensor system:
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*Received the bacterial strain: pseudomonas KE707.</div>
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=='''Lab work'''==
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===='''Objective : obtaining BBa_K1155000'''====
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*'''A.aero/anaerobic regulation system'''
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**''2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3 ''
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===='''1 - Estimation of Pndh* Colony PCR size fragments'''====
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**''BioBrick RBS+amilCP+terminator in plasmid PSB1C3 ''
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Abdou, Anaïs, Sheng
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We used software gene manager to find the correct size of our fragments.
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Estimated size :
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* VF/VR primer -> Plasmid without Pndh* size : 277bp
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* VF/Pfnr_down -> Plasmid with Pndh* size : 276bp
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* Pfnr_Up/VR -> Plasmid with Pndh* size : 311bp
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===='''2 - Electrophoresis of Colony PCR products'''====
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Zhou
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{|
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| style="width:350px;border:1px solid black;" |[[File:Psgel0407.jpg]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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* Well 1 to 4 : 5µL of PCR products with VF/VR primers+1µL of 6X loading dye
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* Well 5 to 6 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
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* Well 7 : 6µL of DNA Ladder
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* Well 8 to 9 : 5µL of PCR product withVF/Pfnr_Down primers+1µL of 6X loading dye
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* Well 10 to 13 : 5µL of PCR products with Pfnr_Up/VR primers+1µL of 6X loading dye
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* Well 14 : 6µL of DNA Ladder
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* Gel : 1.5%
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|}
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{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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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.
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|}
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===='''3 - Stock of BBa_K1155000'''====
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 +
Zhou
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Used quantities :
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* BBa_K1155000 confirmed : 1 mL
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* Glycerol : 500µL glycerol.
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We stocked them at -20°C.
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===='''4 - Extraction of BBa_K1155000 from DH5α'''====
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Anaïs, Sheng
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Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
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<u>Electrophoresis band size estimation</u>
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===='''5 - Digestion of BBa_K1155000 by NotI, MluI, HpaI to check good insertion of Pndh* in pSB1C3'''====
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<p>We used Clonemanager for band size estimation:</p>
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<br>
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{| border="1" align="center"
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|-
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|molecule
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|Primer pair
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|Size
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|-
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|Plasmid without fnr
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|VF/VR
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|277bp
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|-
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|Plasmid with fnr
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|Pfnr_up/Pfnr_down
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|276bp
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|-
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|Plasmid with fnr
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|Pfnr_up/VR
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|311bp
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|}<br>
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Abdou
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<u>PCR interpretation</u><br>
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Used quantities :
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* BBa_K1155000 : 2µL
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* Bufer oranger : 2µL
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* NotI or MluI or HpaI : 0.5µL
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* H2O : 15.5µL
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{| align="center"
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We let the digestion 1h30 at 37°C.
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| style="width:350px;border:1px solid black;" | [[File:PCRPS040713.jpg|right|350px]]
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| style="width:350px;border:1px solid black;" |
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*Well 1,2,5,6,10,11 : plasmid without fnr
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*Well 3,4,8,9,12,13 : plasmid with fnr
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*Well 1 to 4 : primer vf/vr
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*Well 5,6,,8,9 : primer vf/fnr_down
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*Well 10 to 13 : primer fnr_up/vr
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*gel 1.5%
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|}<br>
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<p>We observed for well 10 and well 11 there were a problem which could be some fault in the manipulation. However, the well 4,9,13 conformed to our estimation.</p>
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===='''6 - Culture of BBa_K1155000'''====
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<br>
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<u>Stock BBa_K1155000</u><br>
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<p>1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.</p><br>
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<u>Plasmid DNA extraction</u><br>
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From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.<br>
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<u>Restriction digest</u><br>
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<p>We used 3 enzymes of restriction, they were Not I, Mlu I and Hpa I. And we prepared 2 * 3 = 6 tubes.
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So we add into each tube:</p>
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*Extracted DNA solution : 2µl
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*Buffer Oranger : 2µl
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*Enzyme : 0.5µl
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*H2O : 15.5µl
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*Total : 20µl
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<p>The incubation was at 37°C during 90min</p>
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Anaïs, Sheng
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We made 2 cultures of bacterias transformed with BBa_K1155000 that show a fragments at the right size at electrophoresis.
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<br>
 
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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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