Team:Warsaw/Calendar-Main/18 August 2008

From 2008.igem.org

(Difference between revisions)
(New page: {{WarNotebook}} <!-- do not edit above me! --> <h3>Cloning of protein A DNA to pET15b+OmpA-alfa plasmid in place of OmpA<br>Antoni</h3> <li>Inoculated to liquid LB + ampicilin pGeneart...)
 
(17 intermediate revisions not shown)
Line 2: Line 2:
<!-- do not edit above me! -->
<!-- do not edit above me! -->
 +
<html>
 +
<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
 +
<p>Measurement of concentration of proteins (<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#bca">BCA method</a>). </p>
-
<h3>Cloning of protein A DNA to pET15b+OmpA-alfa plasmid in place of OmpA<br>Antoni</h3>
 
-
<li>Inoculated to liquid LB + ampicilin pGeneart+Z and pKS+A</li>
 
 +
<h3>Cloning of protein A DNA to GeneArt plasmid</h3><h4>Antoni</h4>
 +
<p>Inoculation to liquid LB + ampicilin with bacteria carrying <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneart+Z</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS+A</a> plasmids.</p>
 +
<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></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=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> plasmid using
 +
 +
<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#AlphaPlinkSac">AlphaPlinkSac</a>
 +
 +
primers (gradient of annealing temperature from 55&deg;C to 75&deg;C; elongation length 60s; 35 cycles).</li>
 +
<li>Gel electrophoresis and chioce of proper PCR conditions.</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 using
 +
 +
<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#AlphaPlinkSac">AlphaPlinkSac</a>
 +
 +
primers (annealing temperature - 65&deg;C; elongation length 60s; 20 and 25 cycles (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_August_2008#fig1">Fig. 1.</a>)).</li>
 +
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 550 bp band.</li>
 +
<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated PCR product with SacI (SacI buffer).</li></ol>
 +
 +
 +
 +
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/0/07/Go20.jpg" width=300/></a> <var><b>Fig. 1.</b>PCR to obtain alpha with various number of cycles.<br>
 +
1. Marker<br>
 +
2. Alpha PCR, 25 cycles <br>
 +
3. Alpha PCR, 20 cycles <br></var>
 +
 +
</html>
<!-- do not remove this! -->
<!-- do not remove this! -->
{{WarNotebookEnd}}
{{WarNotebookEnd}}

Latest revision as of 19:02, 26 October 2008

Gallery Bricks Notebook Team Project Home


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

 


Purification of proteins: Z-alpha and Z-omega

Piotr

Measurement of concentration of proteins (BCA method).

Cloning of protein A DNA to GeneArt plasmid

Antoni

Inoculation to liquid LB + ampicilin with bacteria carrying pGeneart+Z and pKS+A plasmids.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. PCR on pUC19 plasmid using AlphaL+SacI and AlphaPlinkSac primers (gradient of annealing temperature from 55°C to 75°C; elongation length 60s; 35 cycles).
  2. Gel electrophoresis and chioce of proper PCR conditions.
  3. PCR on pUC19 plasmid using AlphaL+SacI and AlphaPlinkSac primers (annealing temperature - 65°C; elongation length 60s; 20 and 25 cycles (Fig. 1.)).
  4. Gel electrophoresis and gel-out of 550 bp band.
  5. Overnight digest of isolated PCR product with SacI (SacI buffer).
Fig. 1.PCR to obtain alpha with various number of cycles.
1. Marker
2. Alpha PCR, 25 cycles
3. Alpha PCR, 20 cycles

Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/18_August_2008"