Team:Warsaw/Calendar-Main/4 August 2008

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<h3>Preparing pACYC177+OmpA_omega_deltaA construct<h4>Michał K.</h4>
<h3>Preparing pACYC177+OmpA_omega_deltaA construct<h4>Michał K.</h4>
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<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of pACYC177+OmpA_omega_deltaA_alpha (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends blunting with Klenow fragment (3 hr). </li>
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<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of pACYC177+OmpA_omega_deltaA_alpha (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> blunting with Klenow fragment (3 hr). </li>
<li>Gel elctrophoresis 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>Gel elctrophoresis 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><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA fragment (1 hr). </li>
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA fragment (1 hr). </li>

Revision as of 12:45, 12 October 2008

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Cloning of truncated fragment of protein A

Piotr

Inoculation of some pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA colonies of tranformnts.

Checking the expression of omp_omega_A_alpha and omp_A_alpha

Piotr

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

Checking if omp_omega_A_alpha gives ampicillin resistance

Piotr

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

Preparing pACYC177+OmpA_omega_deltaA construct

Michał K.

  1. Digest of pACYC177+OmpA_omega_deltaA_alpha (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends blunting blunting with Klenow fragment (3 hr).
  2. Gel elctrophoresis and gel-out of proper band - 4300 bp.
  3. Ligation of isolated DNA fragment (1 hr).
  4. Transformation of E. coli TOP10 strain with ligation.
  5. Transformants plating on LB + kanamycin.