Team:Warsaw/Calendar-Main/28 August 2009

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(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3><div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div></h3> <h4>Kamil</h4> <br /> <p>Tasks:<...)
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<p>Methods:</p>
<p>Methods:</p>
<ul>
<ul>
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<li>The digest mix was prepared as follows:<p><div align=center><pre>20&mu;l plasmid isolation<br/>3&mu;l Buffer O (Fermentas)<br/>1&mu;l EcoRI enzyme<br/>1&mu;l PstI enzyme<br/>5&mu;l H<sub>2</sub>O</pre></li>
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<li>The digest mix was prepared as follows:<p><div align=center><pre>20&mu;l plasmid isolation<br/>3&mu;l Buffer O (Fermentas)<br/>1&mu;l EcoRI enzyme<br/>1&mu;l PstI enzyme<br/>5&mu;l H<sub>2</sub>O</pre></li></ul>
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<br />
</html>
</html>
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<html>
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<h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3>
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<h4>Marcin</h4>
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<br/>
 +
<p>Task 1:</p>
 +
<ul>
 +
<li>Gel-outs of following constructs digested on <a href:"https://2009.igem.org/Team:Warsaw/Calendar-Main/27_August_2009">17.08.09</a>:</li><ul>
 +
<li><a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li>
 +
<li><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> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li>
 +
</ul></ul>
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<p>Methods:</p><ul>
 +
<li>All gel-outs were performed using the EurX gel-out kit according to the manual</a></li></ul>
 +
<p>Results:</p>
 +
<ul><li>Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation</ul></li></br>
 +
<b><p>Comment:</b></p>
 +
<p>Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid
 +
was insufficient to perform gel-out. It is obligatory to digest this construct one more time</p>
 +
<br/>
 +
<p>Task 2:</p>
 +
<ul><li>Digestion of following construct:</li></ul>
 +
<ul><li><a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li>
 +
<p>Methods:</p>
 +
<ul><li>Reaction mixture composition:</li><pre>
 +
20 &mu;l purified plasmid DNA product
 +
1 &mu;l XbaI (Fermentas)
 +
1 &mu;l PstI (Fermentas)
 +
2 &mu;l Buffer Tango (Fermentas)
 +
15 &mu;l MQ water</pre>
 +
<li>Digest was performed about six hours and subsequently thermal inactivated</li></ul>
 +
<p>Task 3:</p>
 +
<ul>
 +
<li>Gel-outs of construct described in Task 2</li>
 +
</ul>
 +
<p>Methods:</p><ul>
 +
<li>All gel-outs were performed using the EurX gel-out kit according to the manual</a></li></ul>
 +
<p>Results:</p>
 +
<ul><li>Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation</ul></li></br>
 +
<b><p>Comment:</b></p>
 +
<p>Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid
 +
may be insufficient for efficient gel-out. Although I decided to continue the task</p>
 +
<br/>
 +
<p>Task 4:</p><ul><li>Cloning <a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> ligated with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> vector</ul></li>
 +
<br/>
 +
<p>Methods:</p>
 +
<ul>
 +
<li>
 +
Ligation mixtures composition:</li><pre>
 +
30 &mu;l digested insert
 +
10 &mu;l digested vector
 +
5 &mu;l Tango buffer(Fermentas)
 +
3 &mu;l dNTPs mixture (EurX, concentration 5 mM)
 +
2 &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/>
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</html>
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{{WarNotebookEnd}}
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Revision as of 23:30, 3 September 2009


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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Amplification of the mgtc promoter using new primers
  • pSB1A3 plasmid digest

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 Salmonella matrix
     29 μl H2O
  • PCR programme: 

     4min 95°C 
    (30s 95°C, 35s 48°C, 40s 72°C)x28
     (30s 95°C, 35s 58°C, 40s 72°C)x28 
     10min 72°C 
     ~ 4°C
  • The PCR results were visualised with gel electrophoresis on 1% agarose gel.
  • The digest mix was prepared as follows:

    60μl plasmid isolation
    7μl Buffer O (Fermentas)
    1μl EcoRI enzyme
    1μl PstI enzyme
    1μl H2O
  • The digest was carried out in 37°C for 3h and inactivated for 15 min. in 80°C.
  • 1μl of CIAP enzyme was added after the inactivation and allowed to work for 1h in 37°C and was later inactivated for 15 min. in 85°C.
  • The resulting mix was separated on 1% agarose gel alongside the PCR and then selected bands were extracted for purification.

Results:

  • No gel picture, but the PCR worked so great that I decided not to irradiate the gel with UV light more than it was necessary for band extraction.

Conclusions:

  • The temperatures were chosen... wisely.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Cro-box digest

Methods:

  • The digest mix was prepared as follows:

    20μl plasmid isolation
    3μl Buffer O (Fermentas)
    1μl EcoRI enzyme
    1μl PstI enzyme
    5μl H2O

Assembly of endosomal detection operon

Marcin


Task 1:

Methods:

  • All gel-outs were performed using the EurX gel-out kit according to the manual

Results:

  • Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation

Comment:

Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid was insufficient to perform gel-out. It is obligatory to digest this construct one more time


Task 2:

  • Digestion of following construct:
  • BBa_C0051 with BBa_B0032 on pSB1A3 plasmid

    • Methods:

    • Reaction mixture composition:
      •  
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) 
2 μl Buffer Tango (Fermentas)
15 μl MQ water
    • Digest was performed about six hours and subsequently thermal inactivated

    Task 3:

    • Gel-outs of construct described in Task 2

    Methods:

    • All gel-outs were performed using the EurX gel-out kit according to the manual

    Results:

    • Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation

    Comment:

    Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid may be insufficient for efficient gel-out. Although I decided to continue the task


    Task 4:


    Methods:

    • Ligation mixtures composition:
      •  
30 μl digested insert
10 μl digested vector 
5 μl Tango buffer(Fermentas)
3 μl dNTPs mixture (EurX, concentration 5 mM) 
2 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


    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
Retrieved from "http://2009.igem.org/Team:Warsaw/Calendar-Main/28_August_2009"