Team:Warsaw/Calendar-Main/29 September 2008

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

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.
Gel electrophoresis of PCR products and gel-out of proper bands (OmpA-linker-omega-linker - 900 bp).

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

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

Fig. 1. PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.

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

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

Fig. 2. Digest of plasmid pGeneArt-Z 1. Marker 2. pGeneArt-Z digested NdeI/BcuI

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.
Gel electrophoresis of PCR products and gel-out of proper band (BBa_K103018 - 1200 bp). Fig. 3.

Fig. 3.Result of PCR on pLac_OmpA_omega_ 1. Marker 2. PCR product

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

@@ Line 42: / Line 42: @@
 <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegLNde">OmegLNde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 55 &deg;C; elongation length 60s) to obtain omega_linker fragment. </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 band (omega_linker - 350 bp) (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig1">Fig. 1.</a>).</li></ol>
+<img src="https://static.igem.org/mediawiki/2008/e/ef/Go_29_09_2008.jpg"/>
+<var><b>Fig. 1. </b> PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.</var>
 <h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a></h3>