Difference between revisions of "Team:Warwick/Protocols"
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<h4>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h4> | <h4>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h4> | ||
| − | <br>Glass slides were prepared | + | <br>Glass slides were prepared <a href="url">Glass Slide Preperation Protocol</a> by being cleaned and functionalised (with HCl and GOPTS respectively). |
<br>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides. | <br>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides. | ||
<br>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand. | <br>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand. | ||
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<br>We tested the extent to which each of our zinc fingers (zif 268, sZF2, sZF10 and sZF14) proteins were expressed on the surface of our cells by using immunofluorescence microscopy. | <br>We tested the extent to which each of our zinc fingers (zif 268, sZF2, sZF10 and sZF14) proteins were expressed on the surface of our cells by using immunofluorescence microscopy. | ||
<br>To do this, a FLAG tag (predesigned to be within our construct) was fused to the surface display anchor proteins to which our zinc finger proteins are attached. | <br>To do this, a FLAG tag (predesigned to be within our construct) was fused to the surface display anchor proteins to which our zinc finger proteins are attached. | ||
| − | <br>The introduction of an anti-flag antibody, followed by a secondary antibody (a fluorescently labelled anti-mouse antibody) allowed our E. coli cells to be visualised. | + | <br>The introduction of an anti-flag antibody, followed by a secondary antibody (a fluorescently labelled anti-mouse antibody) allowed our E. coli cells to be visualised. <a href="url">Bacterial Immunofluorescence Protocol</a>. |
<br>Expected results: | <br>Expected results: | ||
<br>No / little fluorescence: Zinc finger proteins should not be expressed on the surface of the wild type and uninduced E. coli cells. This means that the primary antibody (and therefore the fluorescent secondary antibody) are unable to bind to the cell surface, so very little (or even no) fluorescence should be seen. Only background fluorescence should be seen. | <br>No / little fluorescence: Zinc finger proteins should not be expressed on the surface of the wild type and uninduced E. coli cells. This means that the primary antibody (and therefore the fluorescent secondary antibody) are unable to bind to the cell surface, so very little (or even no) fluorescence should be seen. Only background fluorescence should be seen. | ||
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<p> | <p> | ||
<H3> Experiment 3: Reciprocal experiment - binding of fluorescently labelled oligonucleotides to immobilised cells </H3> | <H3> Experiment 3: Reciprocal experiment - binding of fluorescently labelled oligonucleotides to immobilised cells </H3> | ||
| − | <br>E. coli cells expressing each of our 4 zinc finger proteins were immobilised onto glass slides | + | <br>E. coli cells expressing each of our 4 zinc finger proteins were immobilised onto glass slides <a href="url">Bacterial Immunofluorescence Protocol</a>. |
<br>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added. | <br>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added. | ||
<br>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. | <br>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. | ||
Revision as of 14:13, 17 September 2015