Team:Warsaw/Calendar-Main/1 October 2008

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primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain pLac_OmpA_omega fragment without EcoRI. </li>
primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain pLac_OmpA_omega fragment without EcoRI. </li>
<li> Gel electrophoresis of PCR product 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 (pLac_Omp_omega_ - 1200 bp).</li>
<li> Gel electrophoresis of PCR product 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 (pLac_Omp_omega_ - 1200 bp).</li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of PCR product with XbaI and PstI (Tango buffer).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of above digest reaction. </li>
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</ol>

Revision as of 11:17, 27 October 2008

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Preparation of vectors for Biobricks

Michał K., Piotr

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
  3. Gel elctrophoresis and gel-out of proper bands: 4500 bp - pSB2K3 and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector).

Preparation of Z(BBa_K103004)

Michał K.

Inoculation of some colonies from pSB1A3+Z(BBa_K103004) plate to LB with ampicillin.

Preparation of pLac_OmpA_omega

Michał K.

  1. Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of pLac_OmpA_omega fragment. Ligation for 1 hr.
  2. PCR on ligation of pLac_OmpA_omega fragment using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain pLac_OmpA_omega fragment without EcoRI.
  3. Gel electrophoresis of PCR product and gel-out of proper bands (pLac_Omp_omega_ - 1200 bp).
  4. Digest of PCR product with XbaI and PstI (Tango buffer).
  5. Clean-up of above digest reaction.

Preparation of AID

Michał K.

  1. PCR on pMPMT5+AID plasmid using AIDl and AIDP_HindSpeNotPst primers (annealing temperature 58 °C; elongation length 60s) to obtain AID fragment.
  2. Gel electrophoresis of PCR product and gel-out of proper bands (AID - 600 bp).