Team:Paris Saclay/Notebook/July/4

From 2013.igem.org

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{{Team:Paris_Saclay/incl_debut_generique}}
 
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='''Notebook : July 4'''=
 
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===''Summary: ''===
 
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<div>For regulator system:
 
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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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*'''A.aero/anaerobic regulation system'''
 
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**''2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3 ''
 
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**''BioBrick RBS+amilCP+terminator in plasmid PSB1C3 ''
 
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<u>Electrophoresis band size estimation</u><br>
 
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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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<u>PCR interpretation</u><br>
 
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{| align="center"
 
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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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<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><br>
 
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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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<br><p>The incubation was at 37°C during 90min</p>
 
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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.
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WE OBTAIN OUR FIRST BIOBRICK ! We call it Bba_K1155000.
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|}
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===='''2 - 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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* Glycérol : 500µL glycerol.
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We stocked them at -20°C.
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===='''3 - Extraction of Bba_K1155000 from DH5α'''====
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Anaïs, Sheng
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Protocol : [[Team:Paris_Saclay/Protocols/Hight copy plamid extraction|Hight copy plamid extraction]]
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===='''3 - Digestion of Bba_K1155000 by NotI, MluI, HpaI'''====
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Abdou
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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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We let the digestion 1h30 at 37°C.
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Revision as of 16:25, 22 September 2013

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Contents

Notebook : July 4

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155000

1 - Estimation of Pfnr 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 Pfnr size : 277bp
  • VF/Pfnr_down -> Plasmid with Pfnr size : 276bp
  • Pfnr_Up/VR -> Plasmid with Pfnr size : 311bp

1 - 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 OBTAIN OUR FIRST BIOBRICK ! We call it Bba_K1155000.

2 - Stock of Bba_K1155000

Zhou

Used quantities :

  • Bba_K1155000 confirmed : 1 mL
  • Glycérol : 500µL glycerol.

We stocked them at -20°C.

3 - Extraction of Bba_K1155000 from DH5α

Anaïs, Sheng

Protocol : Hight copy plamid extraction

3 - Digestion of Bba_K1155000 by NotI, MluI, HpaI

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.

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