Team:Warsaw/Calendar-Main/24 September 2008

From 2008.igem.org

(Difference between revisions)
 
(20 intermediate revisions not shown)
Line 3: Line 3:
<html>
<html>
 +
 +
<h3>MutD<sub>5</sub> testing</h3>
 +
<h4>Emilia</h4>
 +
<ol><li>
 +
<p>Inoculation MutD<sub>5</sub> from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)
 +
</li><li>6 hours culture</li><li>
 +
Inoculation MutD<sub>5</sub> from the morning to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)</li>
 +
</p>
 +
 +
</ol>
 +
<h3>Mutagenesis of protein A</h3><h4>Paweł</h4>
<h3>Mutagenesis of protein A</h3><h4>Paweł</h4>
Line 9: Line 20:
<li>Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.</li></ol></p>
<li>Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.</li></ol></p>
-
<h3>Preparation of constructs with alpha-A</h3>
+
 
 +
<h3>Preparation of alpha_A construct</h3>
<h4>Antoni</h4>
<h4>Antoni</h4>
<p>
<p>
<ol>
<ol>
-
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pKS plasmid containing protein A with  
+
<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/pKSII+A>pKS plasmid containing protein A</a> with  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>
-
  primers (20 cycles, elongation duration 15 s, annealing temperature 72&deg;C). </li>
+
  primers and 10% DMSO(20 cycles, elongation 40&nbsp;s, annealing temperature 72&deg;C). </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> plasmid with  
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> plasmid with  
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP_XB">AlphaP_XB</a>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a>
-
  primers (20 cycles, elongation duration 45 s, annealing temperature 63&deg;C).
+
  primers (20 cycles, elongation 45&nbsp;s, annealing temperature 63&deg;C).
<br>
<br>
As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR. </li>
As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR. </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 bands (alpha_linker - 572 bp and linker_A - 467 bp ).</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 bands (alpha_linker - 570 bp and linker_A - 470 bp ).</li>
-
<li>Measurment of concentration of both isolated products</li>
+
<li>Measurment of concentration of both isolated products.</li>
</ol></p>
</ol></p>
 +
 +
<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a></h3>
 +
<h4>Piotr</h4>
 +
<ol><li> <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligation of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A</a> DNA fragments. </li>
 +
<li>Plating on LB + ampicillin.</li>
 +
 +
</ol>
 +
</html>
</html>

Latest revision as of 03:46, 29 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
Interactive list of topics
next notebook entry

 


MutD5 testing

Emilia

  1. Inoculation MutD5 from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)

  2. 6 hours culture
  3. Inoculation MutD5 from the morning to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)

Mutagenesis of protein A

Paweł

  1. Results of mutagenesis: no colonies on any plate.
  2. Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.

Preparation of alpha_A construct

Antoni

  1. PCR on pKS plasmid containing protein A with AL+link10+homo2 and AP+NotI primers and 10% DMSO(20 cycles, elongation 40 s, annealing temperature 72°C).
  2. PCR on pUC19 plasmid with AlphaL+SacI and AlphaP+link10+homo2 primers (20 cycles, elongation 45 s, annealing temperature 63°C).
    As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR.
  3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker - 570 bp and linker_A - 470 bp ).
  4. Measurment of concentration of both isolated products.

Preparation of ΔA (BBa_K103003)

Piotr

  1. E. coli TOP10 transformation with overnight ligation of pSB1A3 and ΔA DNA fragments.
  2. Plating on LB + ampicillin.

Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/24_September_2008"