Difference between revisions of "Team:Sheffield/Notebook"
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<div class = "jumbotron"> | <div class = "jumbotron"> | ||
<h2> CLONING OF HEMERYTHRIN </h2> | <h2> CLONING OF HEMERYTHRIN </h2> | ||
| + | <p> <b> <u> Week 1: </b> </u> <br> | ||
| + | -Literature research <br> | ||
| + | -Decided an experimental outline <br> | ||
| + | -In silico design of hemerythrin constructs <br> | ||
| + | -Synthetic genes and primers required for cloning have been ordered <br> | ||
| + | |||
| + | <b> <u> Week 2: </b> </u> <br> | ||
| + | |||
| + | -Make competent cells for the <i> E.coli </i> strains that we used during our project- Top10, JC28 and W3110 <br> | ||
| + | -Tested the efficiency of transformation in these strains <br> | ||
| + | -Prepared buffers, growth media and pouring agar plates for the experiments we were planning to carry out during the following weeks <br> | ||
| + | -Several experiments have been performed in order to monitor the growth of the wild type (W3110) and siderophore-deficient mutant (JC28) <br> | ||
| + | |||
| + | <b> <u> Week 3: </b> </u> <br> | ||
| + | -ensured we had sufficient amounts of plasmid (pSB1C3, pUC18 and pBSKII) for further cloning experiments by transforming them into Top10 competent cells and re-harvesting them (mini-prep experiments) <br> | ||
| + | -characterise the plasmid stocks obtained (Nano Drop and agarose gel)<br> | ||
| + | -characterised the genotype of wild-type (W3110) and mutant (JC28) strains:<br> | ||
| + | <center> -genomic DNA extractions have been performed </center> <br> | ||
| + | <center> - <i>entC</i> gene has been amplified- PCR </center> <br> | ||
| + | <center> -PCR products have been analysed- agarose gel </center> </p> | ||
| + | |||
<h3> Constitutive hemerythrin expression </h3> | <h3> Constitutive hemerythrin expression </h3> | ||
<h3> Overexpression of hemerythrin </h3> | <h3> Overexpression of hemerythrin </h3> | ||
Revision as of 20:48, 18 October 2016
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NOTEBOOK |
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CLONING OF HEMERYTHRIN
Week 1:
-Literature research
-Decided an experimental outline
-In silico design of hemerythrin constructs
-Synthetic genes and primers required for cloning have been ordered
Week 2:
-Make competent cells for the E.coli strains that we used during our project- Top10, JC28 and W3110
-Tested the efficiency of transformation in these strains
-Prepared buffers, growth media and pouring agar plates for the experiments we were planning to carry out during the following weeks
-Several experiments have been performed in order to monitor the growth of the wild type (W3110) and siderophore-deficient mutant (JC28)
Week 3:
-ensured we had sufficient amounts of plasmid (pSB1C3, pUC18 and pBSKII) for further cloning experiments by transforming them into Top10 competent cells and re-harvesting them (mini-prep experiments)
-characterise the plasmid stocks obtained (Nano Drop and agarose gel)
-characterised the genotype of wild-type (W3110) and mutant (JC28) strains:
