Team:Warsaw/Calendar-Main/25 September 2008

From 2008.igem.org

(Difference between revisions)
Line 32: Line 32:
<li>Inoculation of some colonies from plate to LB with ampicillin.</li></ol>
<li>Inoculation of some colonies from plate to LB with ampicillin.</li></ol>
 +
<h3>Preparation of pT7_alpha_link</h3>
 +
<h4>Michał K.</h4>
 +
<ol>
 +
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_deltaA_alpha</a> plasmid using
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+Nde">AlphaL+Nde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a>
 +
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha_link fragment. </li>
 +
<li> Gel electrophoresis of PCR product <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2</a> 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 (alpha_link - 600 bp).</li>
 +
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with NdeI and SacI (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></ol>
Line 54: Line 63:
  primers (annealing temperature 58 &deg;C; elongation length 45s) to obtain omega_link fragment. </li>
  primers (annealing temperature 58 &deg;C; elongation length 45s) to obtain omega_link fragment. </li>
-
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_deltaA_alpha</a> plasmid using
 
-
 
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+Nde">AlphaL+Nde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a>
 
-
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha_link fragment. </li>
 
<li> Gel electrophoresis of PCR products <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2</a> 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 bands (omega_link - 350 bp and alpha_link - 600 bp).</li>
<li> Gel electrophoresis of PCR products <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2</a> 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 bands (omega_link - 350 bp and alpha_link - 600 bp).</li>
-
<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified PCR products: omega_link and  alpha_link with NdeI and SacI (BamHI buffer). </li>
+
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#Digest">digest</a> of purified PCR products: omega_link and  alpha_link with NdeI and SacI (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 products.</li></ol>
<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 products.</li></ol>

Revision as of 00:36, 27 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 


MutD5 testing

Emilia

  1. Isolation of plasmid from culture inoculated on previous day.
  2. preparation of samples to sequencing

Mutagenesis of protein A

Paweł

Treatment of mutageneses as on 23rd September.

Preparation of alpha_A construct

Antoni

  1. PCR on alpha_linker and linker_A with AlphaL+SacI and AP+NotI primers and 10% DMSO (30 cycles, elongation 60 s, annealing temperature 72°C).
  2. Gel electrophoresis of PCR products and gel-out of proper band (1000 bp).
  3. Measurment of concentration of both isolated products.

Preparation of pSB1A3 carrying ΔA (BBa_K103003)

Piotr, Michał K.

  1. Colony PCR with AL_BNXNE and APSacSpe primers on colonies from plates with transformations pSB1A3 + ΔAFig. 1. No products visible after gel electrophoresis.
  2. Inoculation of some colonies from plate to LB with ampicillin.

Preparation of pT7_alpha_link

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AlphaL+Nde and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha_link fragment.
  2. Gel electrophoresis of PCR product Fig. 2 and gel-out of proper band (alpha_link - 600 bp).
  3. Digest of purified PCR product with NdeI and SacI (BamHI buffer).
  4. Clean-up of digested PCR product.

Preparation of BioBricks

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmegLNde and LinP_BS primers (annealing temperature 58 °C; elongation length 45s) to obtain omega_link fragment.
  2. Gel electrophoresis of PCR products Fig. 2 and gel-out of proper bands (omega_link - 350 bp and alpha_link - 600 bp).
  3. digest of purified PCR products: omega_link and alpha_link with NdeI and SacI (BamHI buffer).
  4. Clean-up of digested PCR products.
Fig. 1. Colony PCR to obtain pSB1A3 + ΔA
1. Marker
2-13. PCR on various colonies
Fig. 2. PCR to obtain
1. Marker
2. alpha_link PCR
3. omega_link PCR