Team:Warsaw/Calendar-Main/13 October 2008

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<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>
<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 (annealing temperature 58 &deg;C; elongation length 60s) to obtain <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> fragment. </li>
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> fragment. </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band 600 bp - <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> PCR product. </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band 600 bp - <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> PCR product. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/13_October_2008#fig1">Fig. 1</a>. </li>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
isolated PCR product <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> with EcoRI and BcuI (BamHI buffer). </li>
isolated PCR product <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> with EcoRI and BcuI (BamHI buffer). </li>
<li>  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. </li>
<li>  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. </li>
<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a>.</li></ol>
<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a>.</li></ol>
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<p class="hide"><img src="https://static.igem.org/mediawiki/2008/0/0a/Go2_13_10_2008.jpg">
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<var><b>Fig. 1.</b>SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha<br>
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1. Marker<br>
 +
2. digested PCR product - alpha_linker<br>
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3. digested PCR product - linker_alpha<br></var></p>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
isolated PCR product (alpha2 fragment) with SacI and BcuI (SacI buffer). </li>
isolated PCR product (alpha2 fragment) with SacI and BcuI (SacI buffer). </li>
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<li>  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. </li>
+
<li>  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/13_October_2008#fig1">Fig. 1</a> </li>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/0/0a/Go2_13_10_2008.jpg"></a>
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/0/0a/Go2_13_10_2008.jpg"></a>
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<var><b>Fig. 1.</b>Go2_13_10_2008 </var>
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<var><b>Fig. 1.</b>SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha<br>
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1. Marker<br>
 +
2. Digested PCR product - alpha_linker<br>
 +
3. Digested PCR product - linker_alpha<br></var>

Latest revision as of 17:59, 28 October 2008

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Preparation of linker_alpha (BBa_K103009)

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlphaPSpe primers (annealing temperature 58 °C; elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp - linker_alpha (BBa_K103009) PCR product. Fig. 1.
  3. Digest of isolated PCR product linker_alpha (BBa_K103009) with EcoRI and BcuI (BamHI buffer).
  4. Clean-up of digested PCR product.
  5. Overnight ligation of DNA fragments: pSB2K3 + linker_alpha (BBa_K103009).

Fig. 1.SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha
1. Marker
2. digested PCR product - alpha_linker
3. digested PCR product - linker_alpha

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

Michał K.

Inoculation of few more colonies from old plate with ligation of pACYC177 + OmpA-linker-omega-linker (BBa_K103016) to liquid LB + kanamycin.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

Inoculation of colonies from plate with ligation of pSB1A3 carrying alpha_linker under PT7 (BBa_K103019) fragment to liquid LB + ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

Inoculation of colonies from plate with ligation of pSB2K3 + omega_linker under PT7 (BBa_K103020) to liquid LB + kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K.

Overnight ligation of DNA fragments: pSB1A3 (from 6 October) + AID under pBAD/araC (BBa_K103002) (from 10 October).

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

  1. Digest of pSB2K3 + OmpA_linker_omega_linker under Plac (BBa_K103018) with BcuI and SacI (SacI buffer), dephosphorylation with CIAP.
  2. PCR on alpha PCR product from 10 October using AlphaL+SacI and LinP_BS2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha2 fragment with proper restriction sites (SacI and BcuI).
  3. Gel electrophoresis and gel-out of proper bands (600 bp for alpha2 PCR product and 3000 bp for pSB2K3 (with OmpA_linker under PLac promoter) fragment.
  4. Digest of isolated PCR product (alpha2 fragment) with SacI and BcuI (SacI buffer).
  5. Clean-up of digested PCR product. Fig. 1
  6. Overnight ligation of DNA fragments: pSB2K3 (with lactose promoter and OmpA_linker) + alpha2.
Fig. 1.SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha
1. Marker
2. Digested PCR product - alpha_linker
3. Digested PCR product - linker_alpha