Team:Warsaw/Calendar-Main/13 August 2009

From 2009.igem.org

(Difference between revisions)
(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3>Amplyfing of Bax sequence</h3> <h4>Justyna</h4> <p>Methods</p> <br/> <ul> <li>PCR reaction mix:</li> <br/> per 50μl: <pre> 5.0 ...)
 
(14 intermediate revisions not shown)
Line 13: Line 13:
per 50μl:
per 50μl:
<pre>
<pre>
-
5.0 μl - 10 x buffer (20mM MgSO4)
+
5.0 μl - 10 x buffer (20mM MgSO<sub>4</sub>)
3.5 μl - dNTP mix (5mM)
3.5 μl - dNTP mix (5mM)
3.0 μl - primer BaxF (0.5 μM)
3.0 μl - primer BaxF (0.5 μM)
3.0 μl - primer BaxR2 (0.5 μM)
3.0 μl - primer BaxR2 (0.5 μM)
-
1.0 μl - plasmid DNA template
+
1.0 μl - template DNA
32.0μl - mQ water
32.0μl - mQ water
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)
</pre>
</pre>
</br>
</br>
-
<li>Template DNA in 3 dilutions - 1x10^1 (pure plasmid DNA), 1x10^2 and 1x10^3.</li>
+
<li>Template DNA in 3 dilutions - 10<sup>1</sup> (pure plasmid DNA), 10<sup>-1</sup> and 10<sup>-2</sup>.</li>
<li>buffer, dNTP mix and polymerase from EURx.</li>
<li>buffer, dNTP mix and polymerase from EURx.</li>
-
 
+
<li>DNA template - human Bax with changed codons to be more efficient in yeast. </li>
-
 
+
<br>
-
</br>
+
<br>
 +
<br>
<p>Thermal cycling conditions for PCR (~7-8 kb):</p>
<p>Thermal cycling conditions for PCR (~7-8 kb):</p>
<br/>
<br/>
Line 47: Line 48:
</br>
</br>
<p>Results:</p>
<p>Results:</p>
-
<li>Best reaction for 1x10^1 and 1x10^2 (more specific), in 69.1°C and 69.8°C.
+
<li>Best reaction for 10<sup>1</sup> and 10<sup>-1</sup> (more specific), in 69.1°C and 69.8°C.
-
<img src="">
+
<br>
 +
<br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2009/e/e2/Baxpcr1.JPG">
 +
</center>
 +
<br>
 +
<br>
 +
<br><br><br>
</ul>
</ul>
</html>
</html>
 +
<html>
 +
<h3><div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div></h3>
 +
<h4>Kamil</h4>
 +
<br />
 +
<p>Tasks:</p>
 +
<ul>
 +
<li>Reamplification of the mgtc promoter</li>
 +
</ul>
 +
<br />
 +
<p>Methods:</p>
 +
<ul>
 +
<li>The PCR mix was prepared as follows:
 +
<p><div align=center><pre>5&mu;l buffer B (EURx)<br/> 2&mu;l 5mM dNTPs (EURx)<br/> 5&mu;l forward starter<br/> 5&mu;l reverse starter<br/> 2&mu;l OptiTaq polymerase (EURx)<br/> 2&mu;l matrix <br/> 29 &mu;l H<sub>2</sub>O </pre></li></ul>
 +
<ul><li>PCR programme:
 +
<p><div align=center><pre> 4min 95&deg;C <br/> (30s 95&deg;C, 35s 58&deg;C, 40s 72&deg;C)x28 <br/> 10min 72&deg;C <br/> ~ 4&deg;C<br/></pre></li></ul>
 +
<ul>
 +
<li>The results were visualised with gel electrophoresis on 1% agarose gel.</li>
 +
<li>The appropriate bands were extracted using Gel-Out kit (A&A Biotechnology) according to the producers protocol.
 +
</ul>
 +
<br />
 +
<p>Results:</p>
 +
<ul>
 +
<li>Gel Electrophoresis:</li>
 +
</ul>
 +
<img src=https://static.igem.org/mediawiki/2009/3/30/2009.07.27.jpg />
 +
<p>From left:
 +
<ol>
 +
<li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li>
 +
<li>50&mu;l of PCR mix</li>
 +
<li>50&mu;l of PCR mix</li>
 +
<li>Negative control</li>
 +
</ol>
 +
<br />
 +
<p>Conclusions:</p>
 +
<ul>
 +
<li>Reamplification was successful.</li>
 +
</ul>
 +
<br />
 +
</html>
 +
 +
<html>
 +
 +
<h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3>
 +
<h4>Marcin</h4>
 +
<br />
 +
<p>Task 1:</p>
 +
<ul><li>Gel-out of samples which was cut out from the gel in <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/12_August_2009">12.08.09</a></li></ul>
 +
<p>Procedure:<p/>
 +
<ul>
 +
<li>Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described <a href="http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf">here</a></li></ul>
 +
<p>Results:<p>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2009/0/01/Gel_outs_13_08_09.png" width="40%" height="40%"></center>
 +
<font face="Times New Roman" size="3"><p><div style="text-align: center;">Evaluation of gel-outs yield</div>
 +
</p></font>
 +
<p><b>Comment:</b></p>
 +
<p>The concentration of DNA is sample containing <a href="http://partsregistry.org/Part:BBa_R0080"><span style="color: black">BBa_R0080</a></span> was surprisingly low (sample 1). Because of this I decided to repeat the digest and try to use another gel-out kit.</p><br/>
 +
<p>Task 2:</p><ul><li>Restriction digest of <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span></li></ul>
 +
<p>Methods:<p/><ul>
 +
<li>Digest of BBa_R0080 using SpeI and PstI</li>
 +
<li>
 +
Reaction mixture composition:</li><pre>
 +
20 &mu;l purified plasmid DNA product
 +
0.5 &mu;l SpeI (Fermentas)
 +
1 &mu;l PstI (Fermentas)
 +
5 &mu;l Buffer Tango (Fermentas)
 +
24 &mu;l MQ water</pre>
 +
</ul>
 +
<br/>
 +
<p>Task 3:</p><ul><li>Cloning the sequences: cro CDS and <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> vector</ul></li>
 +
<br/>
 +
<p>Methods:</p>
 +
<ul>
 +
<li>
 +
Ligation mixtures composition:</li><pre>
 +
15 &mu;l digested cro (or E0022)
 +
8 &mu;l digested vector
 +
3 &mu;l Tango buffer(Fermentas)
 +
3 &mu;l dNTPs mixture (EurX, concentration 5 mM)
 +
1 &mu;l ligase T4 (Fermentas)</pre>
 +
<li>Duration of ligation was about 18 hours; reaction was conducted in 19 &deg;c (approximately).</li>
 +
<li>In the next step ligated samples were thermally inactivated via heating in 80 &deg;c for 20 minutes</li>
 +
</ul>
 +
<br/>
 +
<p>Task 4:</p><ul><li>Prepare the bacterial cultures for isolation of on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid containing:</li>
 +
<ul>
 +
<li>p53</li>
 +
<li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span></li>
 +
<li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span></li>
 +
<li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_E0032"><span style="color: black">BBa_E0032</a></span></li></ul><br/>
 +
</html>
<!-- do not remove this! -->
<!-- do not remove this! -->
{{WarNotebookEnd}}
{{WarNotebookEnd}}

Latest revision as of 20:44, 13 September 2009


Previous day
return to main notebook page
Previous entry
next notebook entry

 



Amplyfing of Bax sequence

Justyna

Methods


  • PCR reaction mix:

    • per 50μl: 
    5.0 μl - 10 x buffer (20mM MgSO4)
3.5 μl - dNTP mix (5mM)
3.0 μl - primer BaxF (0.5 μM)
3.0 μl - primer BaxR2 (0.5 μM)
1.0 μl - template DNA
32.0μl - mQ water
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)

  • Template DNA in 3 dilutions - 101 (pure plasmid DNA), 10-1 and 10-2.
  • buffer, dNTP mix and polymerase from EURx.
  • DNA template - human Bax with changed codons to be more efficient in yeast.



    • Thermal cycling conditions for PCR (~7-8 kb):


    94°C, 1 min 0s

94°C, 0 min 15s
69°C-74°C, 0 min 30s
68°C, 1 min 0s
cycles 1-10

94°C, 0 min 15s
69°C-74°C, 0 min 30s
68°C, 1 min 20s
cycles 11-25

68°C, 7 min 0s
4°C, indefinite

    Results:

  • Best reaction for 101 and 10-1 (more specific), in 69.1°C and 69.8°C.






Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Reamplification of the mgtc promoter

Methods:

  • The PCR mix was prepared as follows: 

    5μl buffer B (EURx)
     2μl 5mM dNTPs (EURx)
     5μl forward starter
     5μl reverse starter
     2μl OptiTaq polymerase (EURx)
     2μl matrix 
     29 μl H2O
  • PCR programme: 

     4min 95°C 
     (30s 95°C, 35s 58°C, 40s 72°C)x28 
     10min 72°C 
     ~ 4°C
  • The results were visualised with gel electrophoresis on 1% agarose gel.
  • The appropriate bands were extracted using Gel-Out kit (A&A Biotechnology) according to the producers protocol.

Results:

  • Gel Electrophoresis:

From left:

  1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
  2. 50μl of PCR mix
  3. 50μl of PCR mix
  4. Negative control

Conclusions:

  • Reamplification was successful.

Assembly of endosomal detection operon

Marcin


Task 1:

  • Gel-out of samples which was cut out from the gel in 12.08.09

Procedure:

  • Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here

Results:

Evaluation of gel-outs yield

Comment:

The concentration of DNA is sample containing BBa_R0080 was surprisingly low (sample 1). Because of this I decided to repeat the digest and try to use another gel-out kit.


Task 2:

Methods:

  • Digest of BBa_R0080 using SpeI and PstI
  • Reaction mixture composition:
    •  
20 μl purified plasmid DNA product
0.5 μl SpeI (Fermentas)
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Task 3:


Methods:

  • Ligation mixtures composition:
    •  
15 μl digested cro (or E0022) 
8 μl digested vector 
3 μl Tango buffer(Fermentas)
3 μl dNTPs mixture (EurX, concentration 5 mM) 
1 μl ligase T4 (Fermentas)
  • Duration of ligation was about 18 hours; reaction was conducted in 19 °c (approximately).
  • In the next step ligated samples were thermally inactivated via heating in 80 °c for 20 minutes

Task 4:

Retrieved from "http://2009.igem.org/Team:Warsaw/Calendar-Main/13_August_2009"