Team:Paris Saclay/Notebook/July/4

From 2013.igem.org

Revision as of 16:09, 22 September 2013 by Nad' (Talk | contribs)

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 4

Summary:

For regulator system:
  • 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






Navigation Home Team Project Labwork Notebook Human practices

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%


Previous day Back to calendar Next day

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/July/4"