Team:Warsaw/Calendar-Main/4 August 2008

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<h3> Cloning of truncated fragment of protein A</h3>  
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<h3> Cloning of truncated fragment of protein A (&Delta;A)</h3>  
<h4>Piotr</h4>
<h4>Piotr</h4>
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<p>Inoculation of  some <a href=phttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>ACYC177+OmpA_alpha + &Delta;A</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + &Delta;A</a> colonies of tranformants.</p>
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<p>Inoculation of  some <a href=phttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYC177+OmpA_alpha + &Delta;A</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + &Delta;A</a> colonies of tranformants.</p>
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<h3>Checking the expression of <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-A-alpha>OmpA_A_alpha</a></h3><h4>Piotr</h4>
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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> expression</h3><h4>Piotr</h4>
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<p>Inoculation of <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-A-alpha>OmpA_A_alpha</a> with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.</p>
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<p>Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.</p>
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<h3>Preparing pACYC177+OmpA_omega_&Delta;A construct</h3><h4>Michał K.</h4>
<h3>Preparing pACYC177+OmpA_omega_&Delta;A construct</h3><h4>Michał K.</h4>
<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment (3 hr). </li>
<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment (3 hr). </li>
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<li>Gel elctrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/2_August_2008#fig1">Fig. 1</a>)and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 4300 bp. </li>
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<li>Gel elctrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/4_August_2008#fig1">Fig. 1</a>) and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 4300 bp. </li>
<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragment. </li>
<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragment. </li>
</ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/1/17/Gucio.jpg" width=240/></a> <var><b>Fig. 1. Gel-out of pACYC177_OmpA_omega_deltaA_alpha digested with NotI/BamHI after blunting ends.</b><br>  
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/1/17/Gucio.jpg" width=240/></a> <var><b>Fig. 1. </b>Gel-out of pACYC177_OmpA_omega_deltaA_alpha digested with NotI/BamHI after blunting ends.<br>  
1. Marker<br>
1. Marker<br>
2. pACYC177_OmpA_omega_deltaA_alpha <br></var>
2. pACYC177_OmpA_omega_deltaA_alpha <br></var>

Latest revision as of 10:50, 29 October 2008

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Cloning of truncated fragment of protein A (ΔA)

Piotr

Inoculation of some pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA colonies of tranformants.

Checking OmpA_omega_ΔA_alpha expression

Piotr

Inoculation of OmpA_omega_ΔA_alpha with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Emilia

Inoculation of OmpA_omega_ΔA_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL.

Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

  1. Digest of pACYC177+OmpA_omega_ΔA_alpha (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends blunting with Klenow fragment (3 hr).
  2. Gel elctrophoresis (Fig. 1) and gel-out of proper band - 4300 bp.
  3. Overnight ligation of isolated DNA fragment.
Fig. 1. Gel-out of pACYC177_OmpA_omega_deltaA_alpha digested with NotI/BamHI after blunting ends.
1. Marker
2. pACYC177_OmpA_omega_deltaA_alpha


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