Team:Warsaw/Calendar-Main/16 May 2008

From 2008.igem.org

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<html><h3>PCRs for fusions</h3>
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<html><h3>PCRs for fusions<br>
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<h4>Michał K.:</h4>
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Michał K.</h3>
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<p>
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<ol><li>Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/15_May_2008">15 May</a> PCR's) and DNA <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).<p>
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<ol><li>Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/15_May_2008">15 May</a> PCR's) and DNA <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).</li>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60&deg;C - 80&deg;C).</p>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60&deg;C - 80&deg;C).
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Primers: <br>
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<p>Primers: </p>
 
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<p>
 
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a> <br>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a> <br>
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35 cycles <br>
35 cycles <br>
</li>
</li>
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<li> Optimization of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase - MgCl<sub>2</sub> concentration and number of cycles.
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<li> Optimization of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase - MgCl<sub>2</sub> concentration and number of cycles.<br>
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<p>Primers: </p>
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Primers: <br>
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<p>
 
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and  
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a></p>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a><br>
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</ol></p>
</ol></p>
<img src="https://static.igem.org/mediawiki/2008/f/f9/Pcr_aid_t7_WAW1.jpg"width=350/>
<img src="https://static.igem.org/mediawiki/2008/f/f9/Pcr_aid_t7_WAW1.jpg"width=350/>
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<h3>PCR products - optimalization of annealing temperature: 1-DNA ladder; 2-annealing temperature 60&deg;C, 8-annealing temperature 80&deg;C</h3>
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<var>PCR products - optimalization of annealing temperature: 1-DNA ladder; <br>2-annealing temperature 60&deg;C, 8-annealing temperature 80&deg;C</var>
<img src="https://static.igem.org/mediawiki/2008/d/d1/Pcr_aid_t7_WAW2.jpg"width=350/>
<img src="https://static.igem.org/mediawiki/2008/d/d1/Pcr_aid_t7_WAW2.jpg"width=350/>
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<h3>PCR products - optimalization of MgCl<sub>2</sub> concentration and number of cycles: <br>
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<var>PCR products - optimalization of MgCl<sub>2</sub> concentration and number of cycles: <br>
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1-DNA ladder; <br>
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<ol>
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2-2μl MgCl<sub>2</sub>, 20 cycles; <br>
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<li style="font-family: monospace; margin-left: 3cm">
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3-3μl MgCl<sub>2</sub>, 20 cycles; <br>
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DNA ladder; <br>
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4-2μl MgCl<sub>2</sub>, 25 cycles; <br>
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<li style="font-family: monospace; margin-left: 3cm">
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5-3μl MgCl<sub>2</sub>, 25 cycles;<br>
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2 μl MgCl<sub>2</sub>, 20 cycles; </li>
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6-2μl MgCl<sub>2</sub>, 30 cycles;<br>
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<li style="font-family: monospace; margin-left: 3cm">
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7-3μl MgCl<sub>2</sub>, 30 cycles;<br>
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3 μl MgCl<sub>2</sub>, 20 cycles; </li>
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8-2μl MgCl<sub>2</sub>, 35 cycles;<br>
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<li style="font-family: monospace; margin-left: 3cm">
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9-3μl MgCl<sub>2</sub>, 35 cycles;
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2 μl MgCl<sub>2</sub>, 25 cycles; </li>
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</h3>
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<li style="font-family: monospace; margin-left: 3cm">
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3 μl MgCl<sub>2</sub>, 25 cycles; </li>
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<li style="font-family: monospace; margin-left: 3cm">
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2 μl MgCl<sub>2</sub>, 30 cycles; </li>
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<li style="font-family: monospace; margin-left: 3cm">
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3 μl MgCl<sub>2</sub>, 30 cycles; </li>
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<li style="font-family: monospace; margin-left: 3cm">
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2 μl MgCl<sub>2</sub>, 35 cycles; </li>
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<li style="font-family: monospace; margin-left: 3cm">
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3 μl MgCl<sub>2</sub>, 35 cycles;</li>
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</ol></var>
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Revision as of 14:10, 9 October 2008

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PCRs for fusions
Michał K.

  1. Gel electrophoresis (15 May PCR's) and DNA isolation from proper bands (600 bp for AID lane and 2700 bp for T7 polymerase lanes).
  2. Electrophoresis to estimate the concentration of isolated DNA.
  3. PCR - translation fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60°C - 80°C). Primers:
    AIDlNrH and T7pXbSal
    Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion
    Elongation time: 4 minutes
    35 cycles
  4. Optimization of PCR - translation fusion: AID + T7 RNA-polymerase - MgCl2 concentration and number of cycles.
    Primers:
    AIDlNrH and T7pXbSal
    Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion
    Annealing temperature: 73°C
    Elongation time: 4 minutes
  5. Gel electrophoresis of PCR products.

PCR products - optimalization of annealing temperature: 1-DNA ladder;
2-annealing temperature 60°C, 8-annealing temperature 80°C PCR products - optimalization of MgCl2 concentration and number of cycles:
  1. DNA ladder;
  2. 2 μl MgCl2, 20 cycles;
  3. 3 μl MgCl2, 20 cycles;
  4. 2 μl MgCl2, 25 cycles;
  5. 3 μl MgCl2, 25 cycles;
  6. 2 μl MgCl2, 30 cycles;
  7. 3 μl MgCl2, 30 cycles;
  8. 2 μl MgCl2, 35 cycles;
  9. 3 μl MgCl2, 35 cycles;

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