Team:Warsaw/Calendar-Main/7 July 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h3>Preparation of constructs on pET15b: OmpA_alpha and OmpA_omega</h3>
<h4>Piotr, Weronika</h4>
<h4>Piotr, Weronika</h4>
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Revision as of 16:29, 26 October 2008

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Preparation of constructs on pET15b: OmpA_alpha and OmpA_omega

Piotr, Weronika

  1. Digest of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI (Tango 2x buffer).
  2. Digest of pACYC177 plasmid with NdeI and BamHI (Tango 2x buffer), dephosphorylation with CIAP.
  3. Gel electrophoresis of digested plasmids and gel-out of proper bands (OmpA_alpha- 1000 bp, OmpA_omega - 800 bp).
  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


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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