Team:Groningen/protocols/Transformation

From 2013.igem.org

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<div class="mainContent">
<div class="mainContent">
-
<h2>B. subtilis transformation</h2>
+
<h2><i>B. subtilis </i>transformation</h2>
-
<br/>Losick protocol
+
<H5>The Losick protocol is used</H5>
 +
5
 +
Day 0: Streak out the Bacillus strain of use and plate this on an LB agar plate o/n at 37°C.
-
<br/>-1 Day: Streak out strain and incubate plate o/n at 37 °C.
+
<p>Transformation (D-Day):
-
<br/>Transformation (D-Day):
+
<br>1. Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see sub1).
 +
<br>2. Grow at 37°C for 5 hours (longer if the culture is not really turbid).
 +
<br>3. Mix 400 µl of culture with DNA* in a fresh tube (i.e. 15 ml tubes loosely closed – aeration)
 +
<br>4. Grow the cells at 37°C for an additional 2 hours .
 +
<br>5. Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight.<br/>
-
<br/>1. Pick up a nice big colony and drop it in 2 ml of completed MC (1x)(see sub1).
+
<p>
-
<br/>2. Grow at 37 °C for 5 hours (more if culture is not really turbid).
+
(*usually 1 µl. Then 10 µl of Qiagen plasmid miniprep or <1 µl of chromosomal prep)
-
<br/>3. Mix 400 µl of culture with DNA* in fresh tube (i.e. 15 ml tubes loosely closed – aeration)
+
<p>
-
<br>(*usually 1 µl. Then 10 µl of Quiagen plasmid miniprep or <1 µl of chromosal prep)
+
-
<br>4. Grow for an additional 2 hours at 37 °C.
+
-
<br>5. Plate all on selective antibiotic plates, and incubates at 37°C o/n.<br/>
+
 +
<br><H5>Sub1: Competence medium (MC completed)</H5>
 +
<table id="normal" width ="60%">
 +
  <tr>
 +
    <th>compound</th>
 +
    <th>amount</th>
 +
    <th>treatment</th>
 +
  </tr>
-
<br/>Sub1: Copmpetence medium (MC completed)
+
  <tr>
-
<table border="1">
+
    <td>MQ water</td>
-
<tr>
+
    <td>1.8 ml</td>
-
<td>H20</td>
+
    <td></td>
-
<td>1,8 ml</td>
+
  </tr>
-
<td></td>
+
-
</tr>
+
-
<tr>
+
-
<td>10x MC (sub2)</td>
+
-
<td>200 ul</td>
+
-
<td> filter sterilize</td>
+
-
</tr>
+
-
<tr>
+
-
<td>MgSO4</td>
+
-
<td>6,7 ul</td>
+
-
<td>autoclave sterilize</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Tryptophan 1%</td>
+
-
<td>10 ul</td>
+
-
<td>filter steralize, wrap in Al</td>
+
-
</tr></table>
+
-
<br/>Sub2: MC 10x
+
  <tr>
-
<table border="1">
+
    <td>10x MC (Sub2)</td>
-
<tr>
+
    <td>200 &micro;l</td>
-
<td>for</td>
+
    <td>filter sterilized</td>
-
<td>100 ml</td>
+
  </tr>
-
<td>10 ml</td>
+
-
</tr>
+
-
<tr>
+
-
<td>K2H PO4</td>
+
-
<td>14,036g</td>
+
-
<td> 1,4036g</td>
+
-
</tr>
+
-
<tr>
+
-
<td>KH2 PO4</td>
+
-
<td>5,239g</td>
+
-
<td>0,5239g</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Glucose</td>
+
-
<td>20g</td>
+
-
<td>2g</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Tri-Na Citrate 300mM(Sub3)</td>
+
-
<td>10ml</td>
+
-
<td>1ml</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Ferric NH4 citrate(Sub4)</td>
+
-
<td>1ml</td>
+
-
<td>0,1ml</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Casein Hydrolysate</td>
+
-
<td>1g</td>
+
-
<td>0,1g</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Potassium Glutamate</td>
+
-
<td>2g</td>
+
-
<td>0,2g</td>
+
-
</tr>
+
-
</table>
+
-
Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C<br/>
+
 +
  <tr>
 +
    <td>MgSO<sub>4</sub></td>
 +
    <td>6.7 &micro;l</td>
 +
    <td>autoclaved</td>
 +
  </tr>
-
<br/>Sub3: Tri-Na Citrate 300mM
+
  <tr>
-
<table border="1">
+
    <td>Tryptophan 1%</td>
-
<tr>
+
    <td>10 &micro;l</td>
-
<td>Tri-Na Citrate</td>
+
    <td>filter sterilized (stored in <br>aluminium foil)</td>
-
<td>0,8823g</td>
+
  </tr>
-
</tr>
+
 
-
<tr>
+
</table>
-
<td>H2O</td>
+
 
-
<td>10ml</td>
+
<br><p><H5>Sub2: MC 10x</H5>
-
</tr>
+
<table id="normal" width="60%">
-
</table>
+
  <tr>
-
Wrap in aluminium<br/>
+
    <th>compound</th>
 +
    <th>amount 10 ml</th>
 +
    <th>amount 100 ml</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>K<sub>2</sub>HPO<sub>4</sub></td>
 +
    <td>1.40 g</td>
 +
    <td>14.04 g</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>KH<sub>2</sub>PO<sub>4</sub></td>
 +
    <td>0.52 g</td>
 +
    <td>5.24 g</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Glucose</td>
 +
    <td>2 g</td>
 +
    <td>20 g</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Tri-Na Citrate 300 mM <br>(Sub3)</td>
 +
    <td>1 ml</td>
 +
    <td>10 ml</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Ferric NH<sub>4</sub> citrate <br>(Sub4)</td>
 +
    <td>0.1 ml</td>
 +
    <td>1 ml</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Casein Hydrolysate <br></td>
 +
    <td>0.1 g</td>
 +
    <td>1 g</td>
 +
  </tr>
-
<br/>Sub4: Ferric NH4 citrate
 
-
<table border="1">
 
<tr>
<tr>
-
<td>Ferric NH4 citrate</td>
+
    <td>Potassium Glutamate</td>
-
<td>0,22g</td>
+
    <td>0.2 g</td>
-
</tr>
+
    <td>2 g</td>
-
<tr>
+
  </tr>
-
<td>H2O</td>
+
 
-
<td>10ml</td>
+
</table>
-
</tr>
+
 
 +
The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.
 +
 
 +
 
 +
<br><p><H5>Sub3: Tri-Na Citrate 300 mM </H5>
 +
<table id="normal" width="60%">
 +
  <tr>
 +
    <th>compound</th>
 +
    <th>amount</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Tri-Na Citrate</td>
 +
    <td>0.88 g</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>MQ water</td>
 +
    <td>10 ml</td>
 +
  </tr>
 +
 
 +
</table>
 +
Wrap in aluminium foil and store at -20°C.
 +
 
 +
<br><p><H5>Sub4: Ferric NH4 citrate</H5>
 +
<table id="normal" width="60%">
 +
  <tr>
 +
    <th>compound</th>
 +
    <th>amount</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Ferric NH<sub>4</sub></td>
 +
    <td>0.22 g</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>MQ water</td>
 +
    <td>10 ml</td>
 +
  </tr>
</table>
</table>
-
Wrap in aluminium
+
Wrap in aluminium foil and store at -20°C.
</div>
</div>

Latest revision as of 23:42, 4 October 2013

B. subtilis transformation

The Losick protocol is used
5 Day 0: Streak out the Bacillus strain of use and plate this on an LB agar plate o/n at 37°C.

Transformation (D-Day):
1. Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see sub1).
2. Grow at 37°C for 5 hours (longer if the culture is not really turbid).
3. Mix 400 µl of culture with DNA* in a fresh tube (i.e. 15 ml tubes loosely closed – aeration)
4. Grow the cells at 37°C for an additional 2 hours .
5. Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight.

(*usually 1 µl. Then 10 µl of Qiagen plasmid miniprep or <1 µl of chromosomal prep)


Sub1: Competence medium (MC completed)
compound amount treatment
MQ water 1.8 ml
10x MC (Sub2) 200 µl filter sterilized
MgSO4 6.7 µl autoclaved
Tryptophan 1% 10 µl filter sterilized (stored in
aluminium foil)

Sub2: MC 10x
compound amount 10 ml amount 100 ml
K2HPO4 1.40 g 14.04 g
KH2PO4 0.52 g 5.24 g
Glucose 2 g 20 g
Tri-Na Citrate 300 mM
(Sub3)		 1 ml 10 ml
Ferric NH4 citrate
(Sub4)		 0.1 ml 1 ml
Casein Hydrolysate
0.1 g 1 g
Potassium Glutamate 0.2 g 2 g
The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.

Sub3: Tri-Na Citrate 300 mM
compound amount
Tri-Na Citrate 0.88 g
MQ water 10 ml
Wrap in aluminium foil and store at -20°C.

Sub4: Ferric NH4 citrate
compound amount
Ferric NH4 0.22 g
MQ water 10 ml
Wrap in aluminium foil and store at -20°C.