Team:Warsaw/Calendar-Main/16 May 2008

From 2008.igem.org

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<html><h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5+AID-T7</a></h3>
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<h4>Michał K.</h4>
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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).</li>
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Michał K.:
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<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> - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>translational fusion</a>: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60&deg;C - 80&deg;C). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/16_May_2008#fig1">Fig. 1</a>.<br>
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1. PCR - AID for translation fusion
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Primers: <br>
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Primers:
 
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<html>
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<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#AIDlNrH">AIDlNrH</a>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a> <br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpLinB">AIDpLinB</a>
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<br>
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</html>
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Template DNA: pTrc99A-AID
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Annealing temperature: 55 °C
 
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Annealing time: 60 sec
 
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20 cycles
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Template DNA: purified PCR products from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/15_May_2008">15 May</a> - AID and T7 RNA-polymerase for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>translational fusion</a> <br>
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2. PCR - T7 RNA-polimerase for translation fusion
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Elongation time: 4 minutes <br>
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Primers:
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35 cycles <br>
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</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> - translational fusion: AID + T7 RNA-polymerase - MgCl<sub>2</sub> concentration and number of cycles. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/16_May_2008#fig2">Fig. 2</a>.<br>
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Primers: <br>
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<html>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7lLinkB">T7lLinkB</a>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a>
 
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<br>
 
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</html>
 
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Template DNA: E. coli Rosetta genomic DNA
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<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><br>
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Annealing temperature: 62 °C
 
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Annealing time: 4 minutes
 
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20 cycles
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Template DNA: purified PCR products from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/15_May_2008">15 May</a> - AID and T7 RNA-polymerase for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>translational fusion</a> <br>
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3. PCR - T7 RNA-polimerase for transcription fusion
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Annealing temperature: 73&deg;C<br>
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Elongation time: 4 minutes <br>
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</li>
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<li>  Gel electrophoresis of PCR products.</li>
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</ol></p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/f/f9/Pcr_aid_t7_WAW1.jpg"width=350/></a>
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<var><b>Fig. 1.</b> PCR products - optimization of annealing temperature: 1-DNA ladder; <br>2 to 8 -annealing temperature 60&deg;C (in lane 2) to 80&deg;C (in lane 8).</var>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d1/Pcr_aid_t7_WAW2.jpg"width=350/></a>
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<var><b>Fig. 2.</b> PCR products - optimization of MgCl<sub>2</sub> concentration and number of cycles: <br>
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<ol>
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<li style="font-family: monospace; margin-left: 3cm">
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DNA ladder; <br>
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<li style="font-family: monospace; margin-left: 3cm">
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2 μl MgCl<sub>2</sub>, 20 cycles; </li>
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<li style="font-family: monospace; margin-left: 3cm">
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3 μl MgCl<sub>2</sub>, 20 cycles; </li>
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<li style="font-family: monospace; margin-left: 3cm">
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2 μl MgCl<sub>2</sub>, 25 cycles; </li>
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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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Primers:
 
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<html>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7lRBSHi">T7lRBSHi</a>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a>
 
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<br>
 
</html>
</html>
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Template DNA: E. coli Rosetta genomic DNA
 
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Annealing temperature: 62 °C
 
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Annealing time: 4 minutes
 
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20 cycles
 
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Latest revision as of 13:47, 26 October 2008

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Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

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 - translational fusion: AID + T7 RNA-polymerase - optimization (annealing temperature gradient 60°C - 80°C). Fig. 1.
    Primers:
    AIDlNrH and T7pXbSal
    Template DNA: purified PCR products from 15 May - AID and T7 RNA-polymerase for translational fusion
    Elongation time: 4 minutes
    35 cycles
  4. Optimization of PCR - translational fusion: AID + T7 RNA-polymerase - MgCl2 concentration and number of cycles. Fig. 2.
    Primers:
    AIDlNrH and T7pXbSal
    Template DNA: purified PCR products from 15 May - AID and T7 RNA-polymerase for translational fusion
    Annealing temperature: 73°C
    Elongation time: 4 minutes
  5. Gel electrophoresis of PCR products.

Fig. 1. PCR products - optimization of annealing temperature: 1-DNA ladder;
2 to 8 -annealing temperature 60°C (in lane 2) to 80°C (in lane 8). Fig. 2. PCR products - optimization 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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