Team:Paris Saclay/Notebook/July/4

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**''BioBrick RBS+amilCP+terminator in plasmid PSB1C3 ''
**''BioBrick RBS+amilCP+terminator in plasmid PSB1C3 ''
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<u>Electrophoresis band size estimation</u>
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<u>Electrophoresis band size estimation</u><br>
<p>We used Clonemanager for band size estimation:</p>
<p>We used Clonemanager for band size estimation:</p>
<br>
<br>
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<u>Stock BBa_K1155000</u><br>
<u>Stock BBa_K1155000</u><br>
<p>1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.</p><br>
<p>1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.</p><br>
 +
<u>Plasmid DNA extraction</u><br>
<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>
From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.<br>
 +
<u>Restriction digest</u><br>
<u>Restriction digest</u><br>
<p>We used 3 enzymes of restriction, they were Not I, Mlu I and Hpa I. And we prepared 2 * 3 = 6 tubes.
<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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So we add into each tube:</p><br>
*Extracted DNA solution : 2µl
*Extracted DNA solution : 2µl
*Buffer Oranger : 2µl
*Buffer Oranger : 2µl
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*H2O : 15.5µl
*H2O : 15.5µl
*Total : 20µl
*Total : 20µl
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<p>The incubation was at 37°C during 90min</p>
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<br><p>The incubation was at 37°C during 90min</p>

Revision as of 23:16, 18 September 2013

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Notebook : July 4

Summary:

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.

Lab work

  • A.aero/anaerobic regulation system
    • 2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
    • BioBrick RBS+amilCP+terminator in plasmid PSB1C3

Electrophoresis band size estimation

We used Clonemanager for band size estimation:


molecule Primer pair Size
Plasmid without fnr VF/VR 277bp
Plasmid with fnr Pfnr_up/Pfnr_down 276bp
Plasmid with fnr Pfnr_up/VR 311bp


PCR interpretation

PCRPS040713.jpg
  • Well 1,2,5,6,10,11 : plasmid without fnr
  • Well 3,4,8,9,12,13 : plasmid with fnr
  • Well 1 to 4 : primer vf/vr
  • Well 5,6,,8,9 : primer vf/fnr_down
  • Well 10 to 13 : primer fnr_up/vr
  • gel 1.5%

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.


Stock BBa_K1155000

1 ml of the confirmed sample mixed with 500µl glycerol. And we stocked them at -20°C.


Plasmid DNA extraction

From the cell-culture medium(2 samples plasmid with fnr), we performed some plasmid DNA extraction.

Restriction digest

We used 3 enzymes of restriction, they were Not I, Mlu I and Hpa I. And we prepared 2 * 3 = 6 tubes. So we add into each tube:


  • Extracted DNA solution : 2µl
  • Buffer Oranger : 2µl
  • Enzyme : 0.5µl
  • H2O : 15.5µl
  • Total : 20µl

The incubation was at 37°C during 90min




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