From 2008.igem.org
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| - | <h3>Cloning of protein A DNA to pET15b+OmpA-alfa plasmid in place of OmpA<br>Antoni</h3>
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| - | <li> Colony PCR on colonies from plates with transformations pGeneart+A Primers used:
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| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a>
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| - | <li> Confirmed transformant colonies inoculated to liquid LB with ampicillin
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| - | <li>Inoculated to liquid LB with ampicillin pET15b+OmpA-alfa</li>
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Revision as of 23:35, 2 October 2008
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Cloning of protein A DNA to OmpA constructs
- Digest of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)
- Gel electrophoresis and gel-out of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane).
- Ligation of A DNA fragment with both pACYC177 vectors.
- Transformation of E. coli TOP10 strain with ligations.
- Transformants plating on LB + kanamycin.
Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA
Paweł
- Digest of pET15b-OmpA-omega and Z(in GeneArt vector) with NdeI and NotI.
- Gel-out of Z (~200 bp band).
- OvernightLigation of Z into digested pET15b-OmpA-omega.
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