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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