Team:UNITN-Trento/Protocols

From 2013.igem.org

(Difference between revisions)
Line 310: Line 310:
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html>
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html>
-
<span>Prepare the competence medium as follow:</span>
+
<!--biobrick cloning protocol -->
 +
<p>
 +
Prepare the digestion mix as follow:  
 +
</p>
<table>
<table>
-
<tr>
 
-
<th colspan="2">Competence medium (MC completed)</th>
 
-
</tr>
 
<tr>
<tr>
<td>
<td>
-
H2O
+
DNA
</td>
</td>
-
<TD>
 
-
1.8 ml
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
<td>
<td>
-
10x MC
+
500 ng
</td>
</td>
-
<TD>
 
-
200 ul
 
-
</TD>
 
</tr>
</tr>
<tr>
<tr>
<td>
<td>
-
MgSO4 1M
+
10X NEB Buffer
</td>
</td>
-
<TD>
 
-
6.7 ul
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
<td>
<td>
-
trp 1% (for trp - strains)
+
2.5 ul
</td>
</td>
-
<TD>
 
-
10 ul
 
-
</TD>
 
</tr>
</tr>
-
</table>
 
-
 
-
<table>
 
<tr>
<tr>
-
<th colspan="2">
+
<td>
-
MC 10x
+
10X BSA
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="2">
+
-
for 100 ml
+
</td>
</td>
-
</tr>
 
-
<tr>
 
<td>
<td>
-
K2HPO4 3H2O
+
2.5 ul
</td>
</td>
-
<TD>
 
-
14.036 g
 
-
</TD>
 
</tr>
</tr>
<tr>
<tr>
<td>
<td>
-
KH2PO4
+
E1
 +
</td>
 +
<td>
 +
1 ul
</td>
</td>
-
<TD>
 
-
5.239 g
 
-
</TD>
 
</tr>
</tr>
<tr>
<tr>
<td>
<td>
-
Glucose
+
E2
</td>
</td>
-
<TD>
 
-
20 g
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
<td>
<td>
-
Tri-Na Citrate 300 mM
+
1 ul
</td>
</td>
-
<TD>
 
-
10 ml
 
-
</TD>
 
</tr>
</tr>
-
<tr>
+
<tr>
<td>
<td>
-
Ferric NH4 Citrate
+
H2O
 +
</td>
 +
<td>
 +
Up to 25 ul
</td>
</td>
-
<TD>
 
-
1 ml
 
-
</TD>
 
</tr>
</tr>
 +
</table>
 +
 +
<p>
 +
Incubate the reaction mix at 37 &deg;C for 30 min. Disactivate then the enzymes incubating the mix at 80 &deg;C for 20 min.
 +
The next step will be the ligation of the digestion products. The raction mix is prepared as follow:
 +
</p>
 +
<table>
<tr>
<tr>
<td>
<td>
-
Casein Hydrolysate
+
Insert
 +
</td>
 +
<td>
 +
3 fold excess
</td>
</td>
-
<TD>
 
-
1 g
 
-
</TD>
 
</tr>
</tr>
<tr>
<tr>
<td>
<td>
-
K glutamate
+
Vector
</td>
</td>
-
<TD>
+
<td>
-
2 g
+
40 ng
-
</TD>
+
</td>
</tr>
</tr>
<tr>
<tr>
-
<td colspan="2">Mix everything in 40-50 ml H2O, then adjust to 100 ml, filter sterilize, freeze at -20 C</td>
 
-
</tr>
 
-
</table>
 
-
<table>
 
-
<tr>
 
-
<th>
 
-
Tri-Na Citrate 300mM
 
-
</th>
 
<td>
<td>
-
8.823 g
+
10X T4 Ligase Buffer
</td>
</td>
<td>
<td>
-
in 100 ml H2O
+
2 ul
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<th>
 
-
Ferric NH4 citrate
 
-
</th>
 
<td>
<td>
-
2.2 g
+
T4 Ligase
</td>
</td>
<td>
<td>
-
in 100 ml H2O
+
1 ul
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td colspan="3">
+
<td>
-
--> wrap in aluminium foil!!
+
H2O
</td>
</td>
 +
<TD>
 +
Up to 20 ul
 +
</TD>
</tr>
</tr>
 +
</table>
</table>
-
<br/>
+
<p>
-
<ol>
+
Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 &deg;C for 20 min. Transorm 10 ul of the reaction in competent cells.
-
<li>
+
</p>
-
Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see below);
+
-
</li>
+
-
<li>
+
-
Grow at 37 &deg;C for 5 hours (or more if culture is not really turbid);
+
-
</li>
+
-
<li>
+
-
Mix 400 ul of culture with DNA (usually 1 ug) in fresh tube (i.e. 15 ml tubes losely closed);
+
-
</li>
+
-
<li>
+
-
Grow for additional 2 h at 37 &deg;C;
+
-
</li>
+
-
<li>
+
-
Plate all on selective antibiotic plates, and incubate at 37 &deg;C O/N
+
-
</li>
+
-
</ol>
+
</html>|subtilis-transformation}}
</html>|subtilis-transformation}}
Line 615: Line 563:
Prepare your reaction and incubate at RT for 2 hours. Transform half of the reaction into 200μL of “homemade” competent cells (DH5&alpha;, NEB10&beta;, Novablue or other appropriate strains) following a standard transformation protocol. Plate all the cells.
Prepare your reaction and incubate at RT for 2 hours. Transform half of the reaction into 200μL of “homemade” competent cells (DH5&alpha;, NEB10&beta;, Novablue or other appropriate strains) following a standard transformation protocol. Plate all the cells.
</html>|Ligation}}
</html>|Ligation}}
-
 
-
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|BioBrick Cloning|<html>
 
-
<span>Prepare the competence medium as follow:</span>
 
-
<table class="tn-sp-table">
 
-
<tr>
 
-
<th colspan="2">Competence medium (MC completed)</th>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
H2O
 
-
</td>
 
-
<TD>
 
-
1.8 ml
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
10x MC
 
-
</td>
 
-
<TD>
 
-
200 ul
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
MgSO4 1M
 
-
</td>
 
-
<TD>
 
-
6.7 ul
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
trp 1% (for trp - strains)
 
-
</td>
 
-
<TD>
 
-
10 ul
 
-
</TD>
 
-
</tr>
 
-
</table>
 
-
<table class="tn-sp-table">
 
-
<tr>
 
-
<th colspan="2">
 
-
MC 10x
 
-
</th>
 
-
</tr>
 
-
<tr>
 
-
<td colspan="2">
 
-
for 100 ml
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
K2HPO4 3H2O
 
-
</td>
 
-
<TD>
 
-
14.036 g
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
KH2PO4
 
-
</td>
 
-
<TD>
 
-
5.239 g
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
Glucose
 
-
</td>
 
-
<TD>
 
-
20 g
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
Tri-Na Citrate 300 mM
 
-
</td>
 
-
<TD>
 
-
10 ml
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
Ferric NH4 Citrate
 
-
</td>
 
-
<TD>
 
-
1 ml
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
Casein Hydrolysate
 
-
</td>
 
-
<TD>
 
-
1 g
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>
 
-
K glutamate
 
-
</td>
 
-
<TD>
 
-
2 g
 
-
</TD>
 
-
</tr>
 
-
<tr>
 
-
<td>Mix everything in 40-50 ml H2O, then adjust to 100 ml, filter sterilize, freeze at -20 C</td>
 
-
</tr>
 
-
</table>
 
-
<table class="tn-sp-table">
 
-
<tr>
 
-
<th>
 
-
Tri-Na Citrate 300mM
 
-
</th>
 
-
<td>
 
-
8.823 g
 
-
</td>
 
-
<td>
 
-
in 100 ml H2O
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<th>
 
-
Ferric NH4 citrate
 
-
</th>
 
-
<td>
 
-
2.2 g
 
-
</td>
 
-
<td>
 
-
in 100 ml H2O
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td colspan="3">
 
-
--> wrap in aluminium foil!!
 
-
</td>
 
-
</tr>
 
-
</table>
 
-
<br/>
 
-
<ol>
 
-
<li>
 
-
Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see below);
 
-
</li>
 
-
<li>
 
-
Grow at 37 &deg;C for 5 hours (or more if culture is not really turbid);
 
-
</li>
 
-
<li>
 
-
Mix 400 ul of culture with DNA (usually 1 ug) in fresh tube (i.e. 15 ml tubes losely closed);
 
-
</li>
 
-
<li>
 
-
Grow for additional 2 h at 37 &deg;C;
 
-
</li>
 
-
<li>
 
-
Plate all on selective antibiotic plates, and incubate at 37 &deg;C O/N
 
-
</li>
 
-
</ol>
 
-
</html>|biobrick-cloning}}
 
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Digestion|<html>
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Digestion|<html>
Line 827: Line 616:
<h4>Classic Cloning - for plasmids</h4>
<h4>Classic Cloning - for plasmids</h4>
Incubate at 37&deg;C overnight. The day after add 1&micro;L of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37&deg;C.
Incubate at 37&deg;C overnight. The day after add 1&micro;L of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37&deg;C.
-
    <h4>Screening</h4>
+
<h4>Biobricks Cloning</h4>
 +
        Incubate at 37&deg;C for 30 minutes. Then disactivate the enzymes at 80&deg;C for 20 minutes.
 +
        <h4>Screening</h4>
Incubate for 1.5h at 37&deg;C. Run all the digested product on an agarose gel to screen colonies.  
Incubate for 1.5h at 37&deg;C. Run all the digested product on an agarose gel to screen colonies.  
<br/><br/><hr/><br/>
<br/><br/><hr/><br/>

Revision as of 12:23, 31 July 2013