From 2008.igem.org
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| - | <center><h4><p>1. Optimization of PCR to obtain truncated fragment of protein A DNA </center>
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| - | Primers: <html>
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a>
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </html>
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| - | Elongation time: 30s <br>
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| - | - Optimization of annealing temperature (gradient from 55°C to 75°C)<br>
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| - | - Optimization of number of cycles(15, 20, 25, 30, 35)<br>
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| - | 2. PCR to obtain truncated A protein DNA fragment <br>
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| - | <p>Primers:<html>
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a>
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a></html></p>
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| - | Elongation time: 30s <br>
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| - | Annealing temperature: 60°C <br>
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| - | 20 cycles <br>
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| - | 3. Gel electrophoresis and isolation of 250 bp band. <br>
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| - | 4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI. <br>
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| - | 5. Clean-up of digestion reaction. <br>
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| - | 6. Gel electrophoresis for estimation of DNA concentration. <br>
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| - | 7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.
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| | <h4> Michał K.</h4> | | <h4> Michał K.</h4> |
Revision as of 22:45, 11 October 2008
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Michał K.
- Transformation of E. coli TOP10 strain with ligation products (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA).
- Transformants plating on LB + kanamycin.
Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)
Piotr
Test was conducted in E.coli Rosetta strain expressing omp_omega_A_alfa (with and without induction) and omp_A_alfa.
- Spinnign
- Suspending
- Adding of lysis buffer
- Boiling
- Putting into poliacrylamide gel
- Transfer onto nitrocellulose
- Blocking
- Anti-A antibody binding
- Washing
- Anti-rabbit antibody binding
- Developing with BCIP and NBT
[photo of the gel is to be placed here]
We didn't observe differences in espression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by other factor than Lon and OmpT proteases.
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