Team:ULB-Brussels/Materiel&Methods

<html xmlns="http://www.w3.org/1999/xhtml" lang="en">


<img id="logo" src="https://static.igem.org/mediawiki/2012/8/87/Bac.jpg" height="25%" width="25%">
<img id="logo" src="https://static.igem.org/mediawiki/2012/2/21/Dessin_sam.jpg" height="48%" width="48%">
<img id="logo" src="https://static.igem.org/mediawiki/2012/5/55/Terry.jpg" height="25%" width="25%">


</html>

{| style="color:#1b2c8a;background-color:#fff;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
!align="center"|[[Team:ULB-Brussels|Home]]
!align="center"|[[Team:ULB-Brussels/Team|Team]]
!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=ULB-Brussels Official Team Profile]
!align="center"|[[Team:ULB-Brussels/Project|Project]]
<html><center>
<a class="onglet" href="https://2012.igem.org/Team:ULB-Brussels/Introduction">Introduction</a>&nbsp;/&nbsp;
<a class="onglet" href="https://2012.igem.org/Team:ULB-Brussels/Material&Methods">Material & Methods</a>&nbsp;/&nbsp;
<a class="onglet" href="https://2012.igem.org/Team:ULB-Brussels/Results">Results</a>
</center></html>
!align="center"|[[Team:ULB-Brussels/Parts|Parts Submitted to the Registry]]
!align="center"|[[Team:ULB-Brussels/Modeling|Modeling]]
!align="center"|[[Team:ULB-Brussels/Safety|Safety]]
!align="center"|[[Team:ULB-Brussels/Previous|Older wiki's]]
|}



<html xmlns="http://www.w3.org/1999/xhtml" lang="en">	


<br></br>

<center><font color="#000000"; size="100"> Team ULB-Brussels, project page! </font></center>

<balise id="hautdepage"></balise> 

<br></br>





<br></br>

<h1><A NAME="Material & Methods"> Material & Methods </A> </h1>

<table id="toc" class="toc">
<tr>
<td>
<div id="toctitle">
<h2>Sommaire</h2>
</div>
<ul>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#1. E. coli strains"> 1. E. coli strains </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#2. Plasmids"> 2.Plasmids </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#3. Primers"> 3. Primers </A>
<ul><p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#3.1. Primers for first amplification of each gene"> 3.1. Primers for first amplification of each gene </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#3.2. Primers for second amplification"> 3.2. Primers for second amplification </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#3.3. Primers for insertion in immunity pBAD plasmid"> 3.3. Primers for insertion in immunity pBAD plasmid </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#3.4. Primers for fixing BE"> 3.4. Primers for fixing BE </A></ul>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#4. Kits"> 4. Kits </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#5. Restriction"> 5. Restriction </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#6. Ligation"> 6. Ligation </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#7. Preparation of electrocompetent cells"> 7. Preparation of electrocompetent cells </A>
<p><A HREF="https://2012.igem.org/Team:ULB-Brussels/Materiel&amp;Methods#8. Electroporation"> 8. Electroporation </A>

</td>
</tr>
</table>

<font color=black>

<h2><A NAME="1. E. coli strains"> 1. E. coli strains </A> </h2>

<p>&nbsp;&nbsp;MC1061 : F− araD139, Δ(ara-leu)7696,galE15, galK16, Δ(lac)X74, rpsL (Strr), hsdR2 (rK−mK+), mcrA mcrB1 </p>
<p>&nbsp;&nbsp;TOP10 (Invitrogen) : F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 
Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ- </p>

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>

<br></br>

<h2><A NAME="2. Plasmids"> 2.Plasmids </A> </h2>
<p>pP70 : plasmid which contains the microcin C7 operon and a resistance to chloramphenicol</p>
<p>pCID909 : plasmid which contains the microcin B17 operon and a resistance to ampicilin</p>
<p>pBAD18: arabinose-inducible vector containing kanamicyn resistance</p>
<p>pBAD33: arabinose-inducible vector containing chloramphenicol resistance</p>
<p>pSB1K3 : standard iGEM plasmid which has a resistance to Kanamycin </p>
<p>pSB1C3 : standard iGEM plasmid which has a resistance to Chloramphenicol</p> 
<p>pSB1A3 : standard iGEM plasmid which has a resistance to Ampicilin</p>

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>
<br></br>

<h2><A NAME="3. Primers"> 3. Primers </A> </h2>
<p>The following primers were produced by Sigma-Aldrich and the sequences are shown in a 5’-3’ orientation. </p>

<h3><A NAME="3.1. Primers for first amplification of each gene"> 3.1. Primers for first amplification of each gene </A> </h3>


<p>&nbsp;&nbsp;MccBA-FOR</p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGAATTAAAAGCGAGTG-3'
<p>&nbsp;&nbsp;MccBA-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCAGATATGTGAACCACT-3'

<br></br>


<p>&nbsp;&nbsp;	MccBB-FOR</p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGTGCTCCCTGATATTAA-3'
<p>&nbsp;&nbsp;	MccBB-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTATCTCTCCAGACAGCT-3'

<br></br>

<p>&nbsp;&nbsp;	MccBC-FOR </p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGTCAAAACACGAAC-3'
<p>&nbsp;&nbsp;MccBC-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTACTGTAGACATTTATCAT-3'


<br></br>

<p>&nbsp;&nbsp;MccBE-FOR</p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGTTACATTAAAAATGGC-3'
<p>&nbsp;&nbsp;MccBE-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCATTGAGAACTCCAG-3'

<br></br>

<p>&nbsp;&nbsp;MccBF-FOR</p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGACCATACCTCT-3'
<p>&nbsp;&nbsp;MccBF-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCAGTCTCCTGTT-3'

<br></br>


<p>&nbsp;&nbsp;MccCC-FOR</p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGTTAATTGGTGTCTAC-3'
<p>&nbsp;&nbsp;MccCC-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCATACCATCTCCTTTTTAA-3'


<br></br>

<p>&nbsp;&nbsp;MccCF-FOR</p>
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGATGATACAATC-3'
<p>&nbsp;&nbsp;MccCF-REV</p>
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTATTTCTCGGTAG-3'



<br></br>

<h3><A NAME="3.2. Primers for second amplification"> 3.2. Primers for second amplification </A> </h3>
<p>&nbsp;&nbsp;Common-FOR</p>
5'-GCAGAATTCGCGGCCGCTTCTAGAGCCTCAGCTACACGTGCACTG-3'
<p>&nbsp;&nbsp;Common-REV</p>
5'-AGCCTGCAGCGGCCGCTACTAGTAGGTTATAACGCTTGAATTAAGCCGCGCCGCGAAGCGGCGTCGGCTTGAAT
TTATGGGAATGGTAC-3'
<br></br>



<h3><A NAME="3.3. Primers for insertion in immunity pBAD plasmid"> 3.3. Primers for insertion in immunity pBAD plasmid </A> </h3>

<p>&nbsp;&nbsp;ImMccBG-FOR</p>
5'-CATTCTAGAATTAAAGAGGAGAAAATGGATATAATAGAAAAAAG-3'

<p>&nbsp;&nbsp;ImMccBG-REV</p>
5'-GCACATGCATCATCCCCCTAC-3'

<br></br>


<p>&nbsp;&nbsp;ImMccCF-FOR</p>
5'-AGCCAAGCTTGCATGTTATTTCTCGGTAG-3'
<p>&nbsp;&nbsp;ImMccCF-REV</p>
5'-AGCCAAGCTTGCATG TTATGGGAATGGTAC-3'

<br></br>


<p>&nbsp;&nbsp;ImMccCEFOR</p>
5'-ATTCGAGCTCGGTACATGGTGCAGATTATC-3'
<p>&nbsp;&nbsp;ImMccCEREV</p>
5'-TTTCTCCTCTTTAATTTAACCAATTACTTTTGAAT-3'

<br></br>


<h3><A NAME="3.4. Primers for fixing BE"> 3.4. Primers for fixing BE </A> </h3>
<p>&nbsp;&nbsp;MccCB Endo For</p>
5'-TTGCTTTAGGTTCCTTAAGG-3'


<p>&nbsp;&nbsp;MccCB Endo Rev</p>
5'-TCAGCTTCTGGTACCTTATG-3'

<br></br>

<p>&nbsp;&nbsp;MccCB synthgene For1</p>
5'-AAGGATGATTTCTGGAATTCGCGGCCG-3'


<p>&nbsp;&nbsp;MccCB synthgene Rev1</p>
5'-CCTTAAGGAACCTAAAGCAA-3'

<br></br>


<p>&nbsp;&nbsp;MccCB synthgene For2</p>
5'-CATAAGGTACCAGAAGCTGA-3'


<p>&nbsp;&nbsp;MccCB synthgene Rev2</p>
5'-AGCGGCCGCTACTAGTAGGTTATAACGCTT-3'

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>
<br></br>

<h2><A NAME="4. Kits"> 4. Kits </A> </h2>
<p>Gel purification kit:
 Sigma-Aldrich, GenEluteTm PCR Clean-up (ref:NA1020)
<p>Mini prep kit:
 Zymo Research, ZyppyTM Plasmid Miniprep Kit (ref: D4036)

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>
<br></br>

<h2><A NAME="5. Restriction"> 5. Restriction </A> </h2>
<p>•	Run a PCR to amplify the genes of interest with floating restriction sites.
<p>•	Digest the PCR product and the vector by the appropriate restriction enzymes.
<p>&nbsp;&nbsp;&nbsp;&nbsp;	Restriction mix for the PCR products (insert): 
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*Xµl of PCR products (X depending on the concentration of insert of the PCR products).
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*1µl of each restriction enzyme.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*2µl appropriate buffer 10X.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*Add Yµl bi-distilled water to reach a final volume of 20µl.
<p>&nbsp;&nbsp;&nbsp;&nbsp;	Restriction mix for the vector: 
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*200ng of vector.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*1µl of each restriction enzyme.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*2µl appropriate buffer 10X.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*Add Xµl bi-distilled water to reach a final volume of 20µl.
<p>•	Incubate for one hour at 37°C

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>
<br></br>

<h2><A NAME="6. Ligation"> 6. Ligation </A></h2>
<p>•	The ligation reaction proceeds with 100ng of restricted vector. The quantity of the PCR products is calculated according to the following formula: (100ng vector x PCR product size x 3)/(vector size) = PCR product quantity in ng.
<p>&nbsp;&nbsp;&nbsp;&nbsp;	Ligation mix:
<P>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*1µl  T4 DNA ligase.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*2µl buffer (10x).
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*Add the two restrictions reactions in a final volume of 20µl.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*Add bi-distilled water for a final volume of 20µl.
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*ADN
<p>•	Incubate the ligation reaction overnight at room temperature.
<p>•	Electroporate the ligation on bacteria.
<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>
<br></br>

<h2><A NAME="7. Preparation of electrocompetent cells"> 7. Preparation of electrocompetent cells </A></h2>
<p>•	Dilute 100x an overnight culture in LB medium and incubate at 30°C with shaking until the OD600nm reaches 0.5. 
<p>•	Centrifuge in a cold rotor at 4500 rpm 4°C for 30 minutes.
<p>•	Remove supernatant, resuspend in 20ml cold H20, then centrifuge as above. Repeat this step twice.
<p>•	Remove supernatant, resuspend in 10ml cold glycerol, then centrifuge as above.
<p>•	Remove final supernatant carefully, resuspend in 1ml cold glycerol.
<p>•	Store it at -80°C.

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>

<br></br>

<h2><A NAME="8. Electroporation"> 8. Electroporation </A></h2>
<p>&nbsp;&nbsp;&nbsp;DNA dialysis:
<p>•	Fill a petri dish with dH2O.
<p>•	Make float a round 0.025µm Millipore dialysis filter on dH2O.
<p>•	Place the DNA on the Filter for 20 min, then take it back.

<p>&nbsp;&nbsp;&nbsp;Electroporation:
<p>•	Add dialysed DNA in 50µl of electrocompetent cells.
<p>•	Put mixture in an electroporation cuvette.
<p>•	Electroporate it at 2,500 volt, 200Ω and 25µFD.
<p>•	Add quickly 1ml of LB.
<p>•	Put the bacteria for 1 hours at 37°C.
<p>•	Plate the volume you need on dish (with appropriate antibiotics).

<td><a href=""#hautdepage""><img id="logo" src="http://www.clker.com/cliparts/9/2/8/c/1216180855712705788claudita_home_icon.svg.hi.png" height="40px" width="40px" align="right"></a>

<br></br>

<br></br>

<h2></h2>

<br></br>
<br></br>

<center><td><a href="http://www.ulb.ac.be/facs/sciences/index.html"><img id="logo" src="https://static.igem.org/mediawiki/2012/8/87/Logo-sciences.png" height="120px" width="300px"></a>

<td><a href="http://www.ulb.ac.be/"><img id="logo" src="https://static.igem.org/mediawiki/2012/0/0e/Logo-ULB.jpg" height="120px" width="120px"></a>

<td><a href="http://www.ulb.ac.be/inforsciences3/accueil/">
<img id="logo" src="https://static.igem.org/mediawiki/2012/b/b5/Inforsciences.png" height="120px" width="400px"></a>
</center>

</html>