Team:Warsaw/Calendar-Main/30 July 2008

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<h3>Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha</h3><h4>Michał K.</h4>
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<h3>Cloning of omega_A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a></h3><h4>Michał K.</h4>
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<p>Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_A from previous day) inoculated to liquid LB with kanamycin.  
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<p>Separate transformant colonies (tranformation of ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha-A-omega>pACYC177+OmpA_alpha and omega_A</a> from previous day) inoculated to liquid LB with kanamycin.  
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<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 250 bp band. </li>
<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 250 bp band. </li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI (BamHI bauffer). pACYC177 vectors were also <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylated</a>. </li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated PCR product, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega>pACYC177+OmpA_omega</a> with NotI and SacI (BamHI bauffer). pACYC177 vectors were also <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylated</a>. </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digest reaction. </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digest reaction. </li>
<li>Gel electrophoresis for estimation of DNA concentration. </li>
<li>Gel electrophoresis for estimation of DNA concentration. </li>

Revision as of 23:14, 14 October 2008

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Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

Michał K.

Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_A from previous day) inoculated to liquid LB with kanamycin.

Cloning of truncated fragment of protein A

Michał K.

  1. Optimization of PCR to obtain truncated fragment of protein A DNA.
    Primers: AL+SacI AP+NotI
    Elongation time: 30s
    - Optimization of annealing temperature (gradient from 55°C to 75°C)
    - Optimization of number of cycles(15, 20, 25, 30, 35)
  2. PCR to obtain truncated A protein DNA fragment.
    Primers: AL+SacI AP+NotI
    Elongation time: 30s
    Annealing temperature: 60°C
    20 cycles
  3. Gel electrophoresis and gel-out of 250 bp band.
  4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI (BamHI bauffer). pACYC177 vectors were also dephosphorylated.
  5. Clean-up of digest reaction.
  6. Gel electrophoresis for estimation of DNA concentration.
  7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.