Team:Warsaw/Calendar-Main/5 August 2008

From 2008.igem.org

(Difference between revisions)
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<h3>Checking if <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_A_alpha</a> gives ampicillin resistance</h3>  
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<h3>Checking if <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> gives ampicillin resistance</h3>  
<h4>Piotr</h4>
<h4>Piotr</h4>
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<p>Inoculation <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_A_alpha</a> from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.</p>
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<p>Inoculation <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.</p>
<center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
<center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
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Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_A_alpha</a> into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)</p>
Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_A_alpha</a> into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)</p>
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<h3>Checking <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_A_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>OmpA_A_alpha</a> expression</h3>  
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<h3>Checking <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>OmpA_&Delta;A_alpha</a> expression</h3>  
<h4>Piotr</h4>
<h4>Piotr</h4>
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</ol>  
</ol>  
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[a photo of the gel is top be put here]
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[a photo of the gel is to be put here]
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<h3>Preparing pACYC177+OmpA_omega_deltaA construct</h3><h4>Michał K.</h4>
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<h3>Preparing <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYC177+OmpA_omega_&Delta;A</a> construct</h3><h4>Michał K.</h4>
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation. </li>
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation. </li>
<li>Transformants plating on LB + kanamycin. </li></ol>
<li>Transformants plating on LB + kanamycin. </li></ol>

Revision as of 15:19, 25 October 2008

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Cloning of truncated fragment of protein A

Emilia

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest with SacI and BamHI (BamHI buffer).
  3. Gel electrophoresis - proper clones found for both products of ligation.

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr

Inoculation OmpA_omega_ΔA_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Measurement of bacterial culture growth (OD) in the evening:

ampicillin concentration (μg/mL):IPTG concentration (mmol/mL):
00.10.250.50.751
251.5581.4691.5871.491.5661.311
501.4251.4351.5241.0550.9200.935
751.090.9891.4470.9710.9510.992
1000.090.6851.3781.0780.9770.992

Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)

Checking OmpA_omega_ΔA_alpha and OmpA_ΔA_alpha expression

Piotr

  1. Spinning.
  2. Suspending.
  3. Adding of lysis buffer.
  4. Boiling.
  5. Putting into poliacrylamide gel.
  6. Transfer onto nitrocellulose.
  7. Blocking.
  8. Anti-A antibody binding.
  9. Washing.
  10. Anti-rabbit antibody binding.
  11. Developing with BCIP and NBT.
[a photo of the gel is to be put here]

Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

  1. Transformation of E. coli TOP10 strain with ligation.
  2. Transformants plating on LB + kanamycin.


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