Team:Warsaw/Calendar-Main/22 August 2008

From 2008.igem.org

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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> plasmids with NdeI and NotI (Orange buffer). </li>
<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> plasmids with NdeI and NotI (Orange buffer). </li>
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<li>Gel elctrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/21_August_2008#fig1">Fig. 1</a>). Choice of proper clone. </li>
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<li>Gel elctrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/22_August_2008#fig1">Fig. 1</a>). Choice of proper clone. </li>
<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> with NdeI and NotI (Orange buffer). </li>
<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> with NdeI and NotI (Orange buffer). </li>
<li> Gel electrophoresis of digested plasmid and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 470 bp band. </li>
<li> Gel electrophoresis of digested plasmid and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 470 bp band. </li>

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Tests for ampicillin resistance given by protein added to medium

Piotr

Purification of OmpA_alpha from antibiotic resistance contamination:
Transformation of E. coli TOP10 strain with pACTC177+OmpA_alpha.

Cloning of protein A DNA to pET15b+OmpA-alpha plasmid

Antoni

  1. Control digest of pGeneart+A plasmids with NdeI and NotI (Orange buffer).
  2. Gel elctrophoresis (Fig. 1). Choice of proper clone.
  3. Digest of pGeneart+A with NdeI and NotI (Orange buffer).
  4. Gel electrophoresis of digested plasmid and gel-out of 470 bp band.
  5. Overnight ligation of pET15b+alpha and A (pET15b+alpha from 19 August).

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Ligation of digested vectors from 14 August and alpha DNA fragment from 19 August (more vectors added).
  2. Electroporation of E. coli TOP10 with ligations products.
  3. Transformants plating on LB + kanamycin.

Fig. 1.Control digest of freshly ligated pKS+A (lines 1-3). Line 4 - 0.5μg of DNA ladder mix.


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