Team:Warsaw/Calendar-Main/9 October 2008

From 2008.igem.org

(Difference between revisions)
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<h3>Preparation of pT7_omega_link</h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol>
<ol>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_link fragment(from 30 September) (1 hr).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested pET15b vector (from Preparation of vector for pT7 constructs) with <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a>fragment(from <A href=https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008>30 September</a>) (1 hr).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested vector with omega_link (from 30 September) and alpha_link (from 29 September) fragments (1 hr).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on above ligations using
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on above ligations using
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#pETt7L_XNE">pETt7L_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 120s) to obtain pT7_omega_ fragments </li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#pETt7L_XNE">pETt7L_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 120s) to obtain <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> fragments </li>
<li> Gel electrophoresis of PCR products and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (pT7_omega_ - 600 bp).</li>
<li> Gel electrophoresis of PCR products and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (pT7_omega_ - 600 bp).</li>

Revision as of 12:38, 27 October 2008

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Preparation of vector for pT7 constructs

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day - pET15b+OmpA_omega without XbaI.
  2. Control digest of isolated pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found.
  3. Digest of pET15b+OmpA_omega without XbaI plasmid with NdeI and SacI (BamHI buffer), dephosphorylation with CIAP
  4. Gel electrophoresis and gel-out of proper band - 6000 bp.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

  1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with alpha_linker under PT7 (BBa_K103019) fragment(from 25 September) (1 hr).
  2. PCR on above ligation using pETt7L_XNE and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 120s) to obtain alpha_linker under PT7 (BBa_K103019)fragment.
  3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker under PT7 (BBa_K103019) - 800 bp).
  4. Overnight digest of purified PCR product EcoRI and SacI (BamHI buffer).

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_linker under PT7 (BBa_K103020)fragment(from 30 September) (1 hr).
  2. PCR on above ligations using pETt7L_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 120s) to obtain omega_linker under PT7 (BBa_K103020) fragments
  3. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp).
  4. Overnight digest of purified PCR product with EcoRI and BcuI (BamHI buffer).

Preparation of pLac_OmpA_omega

Piotr

  1. Isolation of plasmid from culture inoculated on previous day pSB2K3 + pLac_OmpA_omega (without EcoRI site).
  2. Control digest of isolated pSB2K3 + pLac_OmpA_omega with EcoRI and PstI (Orange buffer) proper clones found.

Preparation of AID

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day pSB1A3+ AID.
  2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found.

Preparation of AraC+pBAD+AID

Piotr

Inoculation of colonies from plate with ligation of pMPMT5+AID without EcoRI site to liquid LB + tetracycline.