http://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&feed=atom&action=historyTeam:CSWProteens/project/Methods - Revision history2024-03-29T00:31:48ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=26738&oldid=prevN oa at 02:21, 21 June 20142014-06-21T02:21:18Z<p></p>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=26724&oldid=prevN oa at 02:20, 21 June 20142014-06-21T02:20:41Z<p></p>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=25706&oldid=prevN oa at 00:25, 21 June 20142014-06-21T00:25:35Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td></tr>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=25704&oldid=prevN oa at 00:25, 21 June 20142014-06-21T00:25:22Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td></tr>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=25702&oldid=prevN oa at 00:25, 21 June 20142014-06-21T00:25:01Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td></tr>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=25699&oldid=prevN oa at 00:24, 21 June 20142014-06-21T00:24:44Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td></tr>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=25690&oldid=prevN oa at 00:23, 21 June 20142014-06-21T00:23:13Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera.</div></td></tr>
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</table>N oahttp://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=25679&oldid=prevBklebe2016 at 00:21, 21 June 20142014-06-21T00:21:19Z<p></p>
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</table>Bklebe2016http://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=21432&oldid=prevBklebe2016 at 03:48, 20 June 20142014-06-20T03:48:27Z<p></p>
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</table>Bklebe2016http://2014hs.igem.org/wiki/index.php?title=Team:CSWProteens/project/Methods&diff=21019&oldid=prevN oa at 23:58, 19 June 20142014-06-19T23:58:28Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This section of our team Wiki gives an overall summary of the various methods we used to construct our PlantiFreeze device. It is important to note that the device is based on a dual plasmid design: one plasmid controlling expression of the recombinant protein, RiAFP, and the other plasmid controlling secretion of the protein. These procedures reflect the revised construction strategy implemented following our discovery that the wrong DNA sequence was originally ordered for synthesis by IDT.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This section of our team Wiki gives an overall summary of the various methods we used to construct our PlantiFreeze device. It is important to note that the device is based on a dual plasmid design: one plasmid controlling expression of the recombinant protein, RiAFP, and the other plasmid controlling secretion of the protein. These procedures reflect the revised construction strategy implemented following our discovery that the wrong DNA sequence was originally ordered for synthesis by IDT.</div></td></tr>
</table>N oa