</td></tr> <tr> <td style="background-color: #EEE"><a href="/wiki/Image:Igem-logo-150px.png" class="image" title="Igem-logo-150px.png"><img alt="" src="/images/7/7b/Igem-logo-150px.png" width="150" height="115" border="0" /></a>Population counter </td><td style="background-color: #F2F2F2" align="center"><img src="/images/9/94/Report.png" border="0" /> <a href="/wiki/IGEM:Paris_Liliane_Bettencourt/Notebook/Paris_2010" title="IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010">Main project page</a>
<img src="/images/c/c3/Resultset_previous.png" border="0" /><a href="/wiki/IGEM:Paris_Liliane_Bettencourt/Notebook/Paris_2010/2010/08/23" title="IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/23">Previous entry</a>      <a href="/wiki/IGEM:Paris_Liliane_Bettencourt/Notebook/Paris_2010/2010/08/25" title="IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/25">Next entry</a><img src="/images/5/5c/Resultset_next.png" border="0" /> </td></tr> <tr>

Aleksandra and Raphaël

Gel extraction (from PCR products)

• WTet-LuxI
• MTet-LuxI
• STet-LuxI

Restriction digest with XbaI and PstI : 15µl gel extraction products + 2x1µl enzyme + 2µl B2 + 0.2µl BSA

• WTet-LuxI
• MTet-LuxI
• STet-LuxI

Restriction digest with XbaI, PstI and AlWNI : 8µl miniprep (13/08) + 3x0,5µl enzyme + 1,5µl B2 + 0.2µl BSA

• WTet-LuxI
• MTet-LuxI
• STet-LuxI

Gel electrophoresis

• WTet-LuxI (PCR product)
• -
• MTet-LuxI (PCR product)
• -
• STet-LuxI (PCR product)
• -
• WTet-LuxI (Triple restriction)
• -
• MTet-LuxI (Triple restriction)
• -
• STet-LuxI (Triple restriction)
• Ladder 1Kb

<a href="/index.php?title=Special:Upload&wpDestFile=Gel2408_1.jpg" class="new" title="Image:Gel2408 1.jpg">Image:Gel2408 1.jpg</a>

Gel extraction

• WTet-LuxI (PCR product)
• MTet-LuxI (PCR product)
• STet-LuxI (PCR product)
• WTet-LuxI (Triple restriction)
• MTet-LuxI (Triple restriction)
• STet-LuxI (Triple restriction)

Ligations

• PmS-attC + WTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PmS-attC + WTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PmS-attC + MTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PmS-attC + MTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PmS-attC + STet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PmS-attC + STet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsS-attC + WTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsS-attC + WTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsS-attC + MTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsS-attC + MTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsS-attC + STet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsS-attC + STet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsM-attC + WTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsM-attC + WTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsM-attC + MTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsM-attC + MTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsM-attC + STet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• PsM-attC + STet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• pLux + LuxI (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
• WLac + LuxI (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)

Controls :

• LuxI (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)
• PsM (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)
• PsS (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)
• PmS (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)

Transformations of DH5α with 5µL of ligation product, plating with 150µl

• PmS-attC + WTet-LuxI (PCR)
• PmS-attC + WTet-LuxI (Triple restr.)
• PmS-attC + MTet-LuxI (PCR)
• PmS-attC + MTet-LuxI (Triple restr.)
• PmS-attC + STet-LuxI (PCR)
• PmS-attC + STet-LuxI (Triple restr.)
• PsS-attC + WTet-LuxI (PCR)
• PsS-attC + WTet-LuxI (Triple restr.)
• PsS-attC + MTet-LuxI (PCR)
• PsS-attC + MTet-LuxI (Triple restr.)
• PsS-attC + STet-LuxI (PCR)
• PsS-attC + STet-LuxI (Triple restr.)
• PsM-attC + WTet-LuxI (PCR)
• PsM-attC + WTet-LuxI (Triple restr.)
• PsM-attC + MTet-LuxI (PCR)
• PsM-attC + MTet-LuxI (Triple restr.)
• PsM-attC + STet-LuxI (PCR)
• PsM-attC + STet-LuxI (Triple restr.)

Controls :

• pLux + LuxI
• WLac + LuxI
• LuxI
• PsM
• PsS
• PmS
• pUC19 (positive control)

Overnight cultures

• 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

Plating glycerols (verification)

• WTet-LuxI
• MTet-LuxI
• STet-LuxI