Team:Warsaw/Calendar-Main/29 September 2008

From 2008.igem.org

(Difference between revisions)
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmLNXNEB">OmLNXNEB</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinPBSNP">LinPBSNP</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmLNXNEB">OmLNXNEB</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinPBSNP">LinPBSNP</a>
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primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain Omp_omega fragment. </li>
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primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain OmpA_omega fragment. </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>

Revision as of 16:42, 24 October 2008

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Preparation of BioBricks

Michał K.

  1. Digest of Z-GeneArt and RFP(????)+deltaA plasmids with NdeI and BcuI (BamHI buffer).
  2. Digest of RFP(????)+deltaA plasmid with NdeI and SacI (BamHI buffer).
  3. Gel electrophoresis of digested plasmids and gel-out of proper bands (Z - 200 bp and both RFP (??????)'s - 2200 bp).
  4. Gel electrophoresis to estimate concentration of purified DNA from previous day.
  5. Overnight ligation of isolated DNA fragments: RFP(??????) + Z and RFP(????) + OmpA_link (from 18 September).
  6. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmLNXNEB and LinPBSNP primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_omega fragment.
  7. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain pLac_OmpA_omega fragment.
  8. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AL_BNXNE and APSacSpe primers (annealing temperature 58 °C; elongation length 45s) to obtain deltaA fragment.
  9. PCR on pACYC177 + OmpA_omega plasmid using LinLSXNE and OmegPSpe primers (annealing temperature 55 °C; elongation length 60s) to obtain link_omega fragment.
  10. Gel electrophoresis of PCR products and gel-out of proper bands (deltaA - 250 bp, pLac_Omp_omega_ - 1200 bp, linker_omega - 350 bp and Omp_omega_ - 900 bp).

Piotr

Inoculation of bacterias received from iGEM HQs ( ).