Team:Warsaw/Calendar-Main/29 September 2008

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<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_Z>pGeneArt-Z</a> and <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> plasmids with NdeI and BcuI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid. (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig2">Fig. 2.</a>)</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_Z>pGeneArt-Z</a> and <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> plasmids with NdeI and BcuI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid. (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig2">Fig. 2.</a>)</li>
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<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/e/e5/Go_Z_25_09_2008.jpgg" width=300/></a><var><b>Fig. 3.</b> Digest of plasmid pGeneArt-Z<br>  
+
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/e/e5/Go_Z_25_09_2008.jpgg" width=300/></a><var><b>Fig. 2.</b> Digest of plasmid pGeneArt-Z<br>  
1. Marker<br>
1. Marker<br>
2. pSB1A3 digested NdeI/BcuI <br></var>
2. pSB1A3 digested NdeI/BcuI <br></var>

Revision as of 20:37, 27 October 2008

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MutD5 testing

Piotr

Sequencing results: there weren't any mutations in plasmids isolated from MutD5 - maybe this strain is too weak as a mutator.

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmLNXNEB and LinPBSNP primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA-linker-omega-linker (BBa_K103016) fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper bands (OmpA-linker-omega-linker - 900 bp).

Preparation of OmpA-linker (BBa_K103006)

Michał K.

  1. Digest of pSB1A3 carrying ΔA (BBa_K103003) plasmid with NdeI and SacI Fig. 3(BamHI buffer). Dephosphorylation (CIAP) of plasmid.
  2. Gel electrophoresis of digested plasmids and gel-out of proper band (pSB1A3 - 2200 bp).
  3. Overnight ligation of isolated DNA fragments: pSB1A3 + OmpA_linker (BBa_K103006) (from 18 September).

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. PCR on pACYC177 + OmpA_omega plasmid using OmegLNde and LinP_BS primers (annealing temperature 55 °C; elongation length 60s) to obtain omega_linker fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper band (omega_linker - 350 bp) (Fig. 1.).

Preparation of Z(BBa_K103004)

Michał K.

  1. Digest of pGeneArt-Z and pSB1A3 carrying ΔA (BBa_K103003) plasmids with NdeI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmid. (Fig. 2.)
  2. Fig. 2. Digest of plasmid pGeneArt-Z
    1. Marker
    2. pSB1A3 digested NdeI/BcuI
  3. Gel electrophoresis of digested plasmids Fig. 3 and gel-out of proper bands (Z - 200 bp and pSB1A3 - 2200 bp).
  4. Overnight ligation of isolated DNA fragments: pSB1A3 + Z (BBa_K103004).

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_linker_omega_linker under Plac (BBa_K103018) fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper band (BBa_K103018 - 1200 bp). Fig. 1.
Fig. 1. PCR on pLac_OmpA_omega_
1. Marker
2. PCR product

Preparation of vectors for Biobricks

Piotr

Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.

Fig. 3. Digests of plasmid pSB1A3
1. Marker
2. pSB1A3 digested NdeI/SacI
3. pSB1A3 digested NdeI/BcuI