Team:Warsaw/Calendar-Main/7 July 2008

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<h3>Preparation of construct pKS with A protein<br>
 
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Michał L., Marcin:</h3>
 
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<p><ol>
 
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<li> Gradient PCR (achieving multiple copies of gene coding A protein):<br>
 
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template DNA pDRIVE-TAPtag - 1 µl<br>
 
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primer
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a></html> - 2 µl<br><html>
 
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primer
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI_N">AL+SacI_N</a> - 2 µl<br>
 
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Pfu buffer with Mg<sup>2+</sup> - 5 µl<br>
 
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10 mM dNTPs - 1 µl<br>
 
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Pfu Turbo polymerase - 0.5 µl<br>
 
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H2O - 38.5 µl<br>
 
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Program:<br>
 
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1. 94&deg;C, 3 min<br>
 
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2. 94&deg;C, 30 sec<br>
 
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3. 62 to 74&deg;C, 45 sec<br>
 
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4. 72&deg;C, 45 sec<br>
 
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5. Repeat of elongation step 25X<br>
 
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6. 72&deg;C, 10 min<br>
 
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7. Hold at 4 &deg;C<br>
 
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</li>
 
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<li> Gel electrophoresis of PCR product.</li>
 
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Isolation</a> of proper band (470 bp) from the gel.</li>
 
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<li> Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated PCR product and pKS vector with NotI and SacI; to the reaction mix with pKS added 0.5 µl of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">CIAP</a>. </li>
 
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Revision as of 14:17, 11 October 2008

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Preparation of constructs with OmpA protein fusions
Piotr:

  1. Digest of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI.
  2. Digest of pACYC177 plasmid with NdeI and BamHI, dephosphorylation with CIAP.
  3. Gel electrophoresis of digested plasmids and gel-out of proper bands.
  4. Electrophoresis of gel-out products.
  5. Overnight ligation of pACYC177 and OmpA_alpha.
  6. Overnight ligation of pACYC177 and OmpA_omega.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

We have just received corrected version of omegaP-link10-homo2 primer. Let's hope this time everything will be fine. We are repeating the PCRs:


PCR
ProductTemplatePrimersProduct length
linker-ApDRIVE-TapTagAL+link10+homo2 and AP+NotI 470 bp
omega-linkerpUC19OmegaL+SacI and OmegaP+link10+homo2400 bp

PCR program for linker-A and omega-linker

TemperatureTime
94°C4:00
94°C0:3028 cycles
gradient 48-55°C0:45
72°C0:50
72°C10:00
4°Cinfinite

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