Team:Warsaw/Calendar-Main/14 July 2008

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<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> 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>PCR on alpha+A PCR products 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#AP+NotI">AP+NotI</a> primers.</li>
<li>PCR on alpha+A PCR products 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#AP+NotI">AP+NotI</a> primers.</li>
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<li>Gel electrophoresis reveal lack of prober 1000 bp band. </li>
+
<li>Gel electrophoresis reveal lack of proper 1000 bp band. </li>
</ol></p>
</ol></p>

Revision as of 18:33, 25 October 2008

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Cloning of protein Z DNA to OmpA constructs

Michał K.

  1. Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (BamHI buffer), pACYC177 was also dephosphorylated with CIAP (3 hr).
  2. Gel electrophoresis and gel-out of proper bands 220 bp (for Geneart_Z lane) and 4050 bp = (pACYC177+OmpA_omega lane).
  3. Ligation of pACYC177+OmpA_omega and Z fragment DNA (1 hr).
  4. Transformation of E. coli TOP10 strain with ligation.
  5. Transformants plating on LB + kanamycin.

Preparation of alpha+A conctruct

Antoni

  1. PCR on pKS plasmid containing protein A with AL+link10+homo2 and AP+NotI primers (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. PCR on alpha+A PCR products with AlphaL+SacI and AP+NotI primers.
  5. Gel electrophoresis reveal lack of proper 1000 bp band.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

We had to start form scratch with this one.

  1. PCR A in 50 µl
    template DNA - pKS-A4 1 µl
    primer AP+NotI - 2 µl
    primer AL+link10+homo2 - 2 µl
    Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
    dNTPs - 1 µl
    Pfu turbo - 0.5 µl
    H2o - 38.5 µl

    Program:
    1. 95°C 3'
    2. 95°C 30"
    3. 62°C 45"
    4. 72°C 45"
    5. 72°C 10'
    6. keeping in 4°C
  2. PCR omega in 50 µl
    template DNA - pUC19 1 µl
    primer OmegaL+SacI - 2 µl
    primer OmegaP+link10+homo2 - 2 µl
    Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
    dNTPs - 1 µl
    Pfu turbo - 0.5 µl
    H2o - 38.5 µl
    Program:
    1. 95°C 3'
    2. 95°C 30"
    3. 62°C 45"
    4. 72°C 45"
    5. 72°C 10'
    6. keeping in 4°C
    25 cycles
  3. Gel electrophoresis
  4. Reisolation from agarose gel