Team:Warsaw/Calendar-Main/10 October 2008

From 2008.igem.org

(Difference between revisions)
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  primers (annealing temperature 45 &deg;C; elongation length 2.30 min) to obtain <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a>fragment. </li>
  primers (annealing temperature 45 &deg;C; elongation length 2.30 min) to obtain <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</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 1800 bp. </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 1800 bp <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_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 with XbaI and PstI (Tango buffer). </li>
isolated PCR product with XbaI and PstI (Tango buffer). </li>
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<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>
<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 (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha fragment. </li>
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha 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. </li>
+
<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="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig1">Fig. 1</a>. </li>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg"></a>
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg"></a>
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<var><b>Fig. 9.</b>Go_10_10_2008.jpg </var>
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<var><b>Fig. 1.</b>Pcr to obtain alpha_linker under PT7 (BBa_K103019), PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha<br>
 +
1. Marker<br>
 +
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)<br>
 +
3. PCR to obtain AID under pBAD/araC (BBa_K103002)<br> 
 +
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha<br></var>
</html>
</html>

Revision as of 17:55, 28 October 2008

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Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K., Piotr

  1. Clean-up of overnight digest reaction.
  2. Digest of pSB1A3 carrying ΔA (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
  3. Gel electrophoresis and gel-out of proper band - 2200 bp.
  4. Ligation of digested pSB1A3 with alpha_linker under PT7 (BBa_K103019) fragment (1 hr).
  5. Transformation of TOP10 with above ligation.
  6. Tranformants plating on LB with ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K., Piotr

  1. Ligation of digested pSB2K3 vector from (30 September) with omega_linker under PT7 (BBa_K103020) fragment (1 hr).
  2. Transformation of TOP10 with above ligation.
  3. Tranformants plating on LB with kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day - pMPMT5+AID (with removed EcoRI site).
  2. Control digest of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.
  3. Temperature gradient PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 40 - 60 °C; elongation length 2.30 min) to optimize conditions for obtaining AID under pBAD/araC (BBa_K103002).
  4. Gel electrophoresis. Best annealing temperature (45 °C) chosen
  5. PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 45 °C; elongation length 2.30 min) to obtain AID under pBAD/araC (BBa_K103002)fragment.
  6. Gel electrophoresis and gel-out of proper band 1800 bp Fig. 1.
  7. Digest of isolated PCR product with XbaI and PstI (Tango buffer).
  8. Clean-up of digested PCR product.
Fig. 13.Grad_arac_09_10_2008.jpg

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AlphaL+SacI and AlphaP+link10+homo2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp Fig. 1.
Fig. 1.Pcr to obtain alpha_linker under PT7 (BBa_K103019), PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha


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