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Team:Warsaw/Calendar-Main/6 October 2008
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<ol><li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> | <ol><li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> | ||
| - | primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> (annealing temperature - 55°C,60 s of elongation step). No visible bands for proper product (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008#fig1">Fig. 1 | + | primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> (annealing temperature - 55°C,60 s of elongation step). No visible bands for proper product (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008#fig1">Fig. 1</a>).</li> |
<li>Inoculation of some colonies which grown on plate - <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> to liquid LB + kanamycin. </li></ol> | <li>Inoculation of some colonies which grown on plate - <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> to liquid LB + kanamycin. </li></ol> | ||
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/66/Kolonijny_alfa.jpg"></a> | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/66/Kolonijny_alfa.jpg"></a> | ||
| - | <var><b>Fig. 1.</b>Colony PCR with LinLSXNE and AlphaPSpe. Lack of expected 400 bp product. | + | <var><b>Fig. 1.</b>Colony PCR with LinLSXNE and AlphaPSpe. Lack of expected 400 bp product.</var> |
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a> | <li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a> | ||
| - | primers on colonies from plates with transformations <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> (annealing temperature - 55°C,90 s of elongation step). No visible bands for proper product (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008#fig2">Fig. 2 | + | primers on colonies from plates with transformations <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> (annealing temperature - 55°C,90 s of elongation step). No visible bands for proper product (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008#fig2">Fig. 2</a>).</li> |
<li>Inoculation of some colonies which grown on plate transformation: <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> to liquid LB + kanamycin. </li></ol> | <li>Inoculation of some colonies which grown on plate transformation: <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> to liquid LB + kanamycin. </li></ol> | ||
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<ol> | <ol> | ||
<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> plasmid with XbaI and PstI (Tango buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li> | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> plasmid with XbaI and PstI (Tango buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li> | ||
| - | <li> Gel electrophoresis | + | <li> Gel electrophoresis 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 - 2200 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008#fig3">Fig. 3</a>.</li> |
</ol> | </ol> | ||
<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/4/49/Go_05_10_2008.jpg"></a> | <a name="fig3"><img src="https://static.igem.org/mediawiki/2008/4/49/Go_05_10_2008.jpg"></a> | ||
| - | <var><b>Fig. 3.</b> | + | <var><b>Fig. 3.</b>XbaI/PstI digest of pSB1A3.<br> |
1. Marker<br> | 1. Marker<br> | ||
| - | 2. pSB1A3<br> </var> | + | 2. XbaI/PstI digest pSB1A3<br> </var> |
Latest revision as of 21:19, 28 October 2008
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Preparation of linker_alpha (BBa_K103009)Michał K.
Fig. 1.Colony PCR with LinLSXNE and AlphaPSpe. Lack of expected 400 bp product.
Preparation of linker_omega (BBa_K103013)Michał K.Inoculation of some colonies which grown on plate - pSB2K3 + linker_omega (BBa_K103013) to liquid LB + kanamycin. Preparation of OmpA-linker-omega-linker (BBa_K103016)Michał K.
Fig. 2.Colony PCR with OmpaL_N and OmpaP_link. Lack of expected 350 bp product.
Preparation of vector for pT7 constructsMichał K.Overnight selfligation of pET15b+OmpA_omega (with removed XbaI). Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)Michał K.Overnight ligation of isolated DNA fragments pSB2K3 (from 1 October) + OmpA_linker_omega_linker under Plac (BBa_K103018) (without EcoRI site). Preparation of AID(BBa_K103001)Michał K.
Fig. 3.XbaI/PstI digest of pSB1A3.1. Marker 2. XbaI/PstI digest pSB1A3
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