Team:Warsaw/Calendar-Main/7 July 2009

From 2009.igem.org

(Difference between revisions)
Line 7: Line 7:
<p>Tasks:</p>
<p>Tasks:</p>
<ul>
<ul>
-
<li>Plasmid assembly</li>
+
<li>Plasmid assembly
</ul>
</ul>
<br />
<br />
Line 16: Line 16:
-
<p>UPDATE: No luck this time, back to the drawing board...
+
<p>UPDATE: No luck this time, back to the drawing board...</p>
 +
</html>
<h3>Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal</h3>
<h3>Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal</h3>
-
<html>
+
 
<h4>Kuba</h4>
<h4>Kuba</h4>
<br />
<br />
Line 29: Line 30:
<ul>
<ul>
<li><p></li>
<li><p></li>
-
 
<br />
<br />
-
<p>Methods:</p>
+
Methods:
<ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul.  
<ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul.  
Line 39: Line 39:
<ul>
<ul>
<li>PCR programs:</li>
<li>PCR programs:</li>
-
<p>hly</p><pre align="left">4min 95&deg;C <br/> (30s 95&deg;C, 40s 41&deg;C, 1min40s 72&deg;C)x3 <br/> (30s 95&deg;C, 40s 44&deg;C, 1min40s 72&deg;C)x28 <br/> 10min 72&deg;C <br/> ~ 7&deg;C</pre>
+
<pre align="middle"> 4min 95&deg;C  
 +
(30s 95&deg;C, 40s 41&deg;C, 1min40s 72&deg;C)x3  
 +
(30s 95&deg;C, 40s 44&deg;C, 1min40s 72&deg;C)x28 10min 72&deg;C  
 +
~ 7&deg;C</pre>
-
<p>random program</p>
 
</ul>
</ul>
<ul>
<ul>
Line 48: Line 50:
<br />
<br />
<p>Results:</p>
<p>Results:</p>
-
<img src="https://static.igem.org/mediawiki/2009/c/cd/Reamplifikacja_llo.JPG"/>
+
https://static.igem.org/mediawiki/2009/c/cd/Reamplifikacja_llo.JPG
<var>
<var>
<ul>
<ul>
Line 63: Line 65:
<p>no siginificant amounts of desired product were obtained
<p>no siginificant amounts of desired product were obtained
-
</html>
 

Revision as of 21:30, 8 July 2009


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

 


Yet another insertion of the mgtc gene into the pKSII+ plasmid

Kamil


Tasks:

  • Plasmid assembly

Methods:

  • The ligation mix was prepared as follows: 1ul plasmid, 3ul gene, 1ul ligation buffer B (Fermentas), 1ul T4 DNA ligase (Fermentas), 1ul 30% PEG, 1ul 10mM ATP, the solution was topped up with H2O to the final volume of 10 ul. The ligation was carried out in 37°C for 1h and then inactivated for 15min. at 65°C.

  • A 200ul batch of chemocompetent bacteria was transformed with 3ul of ligation mix and incubated on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.

  • UPDATE: No luck this time, back to the drawing board...

    Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal

    Kuba


    PCR was preformed on a product of classical biobrick-format Llo (reamplification)


    three reactions were preformed, each with increasing concentration of template

    • </li>
      Methods:

      • <p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul. <p>One of the samples also contained 1 ul of DMSO</p>
      • PCR programs:
      •  4min 95°C 
         (30s 95°C, 40s 41°C, 1min40s 72°C)x3 
         (30s 95°C, 40s 44°C, 1min40s 72°C)x28  10min 72°C 
         ~ 7°C
      • Electrophoretic separation on 1% agarose gel


      <p>Results:

      Reamplifikacja_llo.JPG

      • Gel (from left)
      1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
      2. sample no.1 (1ul of 100-fold diluted template)
      3. sample no. 2 (2ul of 100-fold diluted template)
      4. sample no. 3 (1ul of template)


      5. no siginificant amounts of desired product were obtained

        April
        MTWTFSS
            1 2 3 4 5
        6 7 8 9 10 11 12
        13 14 15 16 17 18 19
        20 21 22 23 24 25 26
        27 28 29 30
        May
        MTWTFSS
                1 2 3
        4 5 6 7 8 9 10
        11 12 13 14 15 16 17
        18 19 20 21 22 23 24
        25 26 27 28 29 30 31
        June
        MTWTFSS
        1 2 3 4 5 6 7
        8 9 10 11 12 13 14
        15 16 17 18 19 20 21
        22 23 24 25 26 27 28
        29 30
        July
        MTWTFSS
            1 2 3 4 5
        6 7 8 9 10 11 12
        13 14 15 16 17 18 19
        20 21 22 23 24 25 26
        27 28 29 30 31
        August
        MTWTFSS
                  1 2
        3 4 5 6 7 8 9
        10 11 12 13 14 15 16
        17 18 19 20 21 22 23
        24 25 26 27 28 29 30
        31
        September
        MTWTFSS
          1 2 3 4 5 6
        7 8 9 10 11 12 13
        14 15 16 17 18 19 20
        21 22 23 24 25 26 27
        28 29 30
        October
        MTWTFSS
              1 2 3 4
        5 6 7 8 9 10 11
        12 13 14 15 16 17 18
        19 20 21 22 23 24 25
        26 27 28 29 30 31