Team:Paris Liliane Bettencourt/Notebook/2010/08/25/

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Aleksandra and Raphaël


Colony PCR (verification of glycerols)


Primers : VF2 + VR

  • 8 colonies from WTet-LuxI glycerol
  • 8 colonies from MTet-LuxI glycerol
  • 8 colonies from STet-LuxI glycerol


Minipreps

  • PsW-attC x2
  • PsM-attc x2
  • PsS-attC x2
  • PmS-attC x2
  • WLac
  • SLac
  • WTet-LuxI x2
  • MTet-LuxI x2
  • STet-LuxI x2
  • pLux
  • LuxI
  • I13507-pLux-GFP


Restriction digest with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 2x1µl enzymes + 0.25µl BSA

  • PsW-attC
  • PsM-attc
  • PsS-attC
  • PmS-attC
  • pLux
  • WLac
  • SLac


Restriction digest with XbaI and PstI : 20µl miniprep + 2.5µl B3 + 2x1µl enzymes + 0.25µl BSA

  • LuxI


Restriction digest with XbaI, PstI and AlwNI : 20µl miniprep + 2.5µl B2 + 3x1µl enzymes + 0.25µl BSA

  • WTet-LuxI
  • MTet-LuxI
  • STet-LuxI


Gel electrophoresis

  • WTet-LuxI colony 1
  • WTet-LuxI colony 2
  • WTet-LuxI colony 3
  • WTet-LuxI colony 4
  • WTet-LuxI colony 5
  • WTet-LuxI colony 6
  • WTet-LuxI colony 7
  • WTet-LuxI colony 8
  • MTet-LuxI colony 1
  • MTet-LuxI colony 2
  • MTet-LuxI colony 3
  • MTet-LuxI colony 4
  • MTet-LuxI colony 5
  • MTet-LuxI colony 6
  • MTet-LuxI colony 7
  • MTet-LuxI colony 8
  • Ladder 1Kb

<a href="/index.php?title=Special:Upload&wpDestFile=Gel2508_1.jpg" class="new" title="Image:Gel2508 1.jpg">Image:Gel2508 1.jpg</a>


Gel electrophoresis

  • STet-LuxI colony 1
  • STet-LuxI colony 2
  • STet-LuxI colony 3
  • STet-LuxI colony 4
  • STet-LuxI colony 5
  • STet-LuxI colony 6
  • STet-LuxI colony 7
  • STet-LuxI colony 8
  • -
  • STet-LuxI (restriction digest)
  • Ladder 1Kb
  • LuxI (restriction digest)

<a href="/index.php?title=Special:Upload&wpDestFile=Gel2508_2.jpg" class="new" title="Image:Gel2508 2.jpg">Image:Gel2508 2.jpg</a>


Gel electrophoresis

  • PsS-attC (restriction digest)
  • -
  • PsM-attC (restriction digest)
  • -
  • PsW-attC (restriction digest)
  • -
  • PmS-attC (restriction digest)
  • -
  • WLac (restriction digest)
  • -
  • SLac (restriction digest)
  • Ladder 1Kb

<a href="/index.php?title=Special:Upload&wpDestFile=Gel2508_3.jpg" class="new" title="Image:Gel2508 3.jpg">Image:Gel2508 3.jpg</a>


Gel electrophoresis

  • WTet-LuxI
  • -
  • MTet-LuxI
  • -
  • Ladder 1Kb
  • -

<a href="/index.php?title=Special:Upload&wpDestFile=Gel2508_4.jpg" class="new" title="Image:Gel2508 4.jpg">Image:Gel2508 4.jpg</a>


Gel extraction

  • PsS-attC
  • PsM-attC
  • PsW-attC
  • PmS-attC
  • WLac
  • SLac
  • WTet-LuxI
  • MTet-LuxI
  • LuxI


PCR purification

  • pLux


Ligations

  • PmS-attC + MTet-LuxI (1,5µl + 3µl + 0.5µl ligase + 1µl buffer + 4µl water)
  • PmS-attC + WTet-LuxI (1,5µl + 6µl + 0.5µl ligase + 1µl buffer + 1µL water)
  • PsM-attC + MTet-LuxI (4µl + 3µl + 0.5µl ligase + 1µl buffer + 1,5µl water)
  • PsM-attC + WTet-LuxI (4µl + 6µl + 0.5µl ligase + 1,1µl buffer)
  • PsW-attC + MTet-LuxI (6µl + 3µl + 0.5µl ligase + 1µl buffer)
  • PsW-attC + WTet-LuxI (6µl + 6µl + 0.5µl ligase + 1,5µl buffer)
  • PsS-attC + MTet-LuxI (1µl + 3µl + 0.5µl ligase + 1µl buffer + 5µl water)
  • PsS-attC + WTet-LuxI (1µl + 6µl + 0.5µl ligase + 1µl buffer + 1,5µl water)
  • pLux + LuxI (1µl + 6µl + 0.5µl ligase + 1µl buffer + 1,5µl water)
  • SLac + LuxI (1µl + 6µl + 0.5µl ligase + 1µl buffer + 1,5µl water)
  • WLac + LuxI (1µl + 6µl + 0.5µl ligase + 1µl buffer + 1,5µl water)


Controls :

  • pLux (1µL + 0.5µl ligase + 1µl buffer + 7,5µl water)
  • SLac (1µL + 0.5µl ligase + 1µl buffer + 7,5µl water)
  • WLac (1µL + 0.5µl ligase + 1µl buffer + 7,5µl water)
  • PmS-attC (1,5µL + 0.5µl ligase + 1µl buffer + 7µl water)
  • PsM-attC (4µL + 0.5µl ligase + 1µl buffer + 4,5µl water)
  • PsW-attC (6µL + 0.5µl ligase + 1µl buffer + 3µl water)
  • PsS-attC (1µL + 0.5µl ligase + 1µl buffer + 7,5µl water)


Overnight cultures

  • WLac-LuxI
  • PsM-MTet I
  • PsM-STet IV
  • PsS-MTet I
  • PsM-WTet I
  • PsM-MTet III
  • PsS-STet IV
  • PsS-WTet III
  • PsM-WTet II
  • PsM-STet II
  • PsS-MTet IV
  • PsS-WTet IV
  • PmS-STet I
  • PmS-WTet I
  • PmS-STet III