Difference between revisions of "Team:Sheffield/Notebook"
| Line 276: | Line 276: | ||
<div class = "jumbotron"> | <div class = "jumbotron"> | ||
<h2> CLONING OF HEMERYTHRIN </h2> | <h2> CLONING OF HEMERYTHRIN </h2> | ||
| − | <p> <b> <u> Week 1: </ | + | |
| + | <p> <b> <u> Week 1:</u> </b> <br> | ||
| + | |||
-Literature research <br> | -Literature research <br> | ||
-Decided an experimental outline <br> | -Decided an experimental outline <br> | ||
| Line 282: | Line 284: | ||
-Synthetic genes and primers required for cloning have been ordered <br> | -Synthetic genes and primers required for cloning have been ordered <br> | ||
| − | <b> <u> Week 2: </ | + | <p> <b> <u> Week 2:</u> </b> <br> |
| − | -Make competent cells for the <i> E.coli </i> | + | -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> | -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> | -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> | -Several experiments have been performed in order to monitor the growth of the wild type (W3110) and siderophore-deficient mutant (JC28) <br> | ||
| − | + | <p> <b> <u> Week 3:</u> </b> <br> | |
| − | <b> <u> Week 3: </ | + | -ensured we had sufficient amounts of plasmid for further cloning experiments: <br> |
| − | - | + | <ul> |
| − | - | + | <li> -transformed the pSB1C3, pUC18 and pBSKII plasmids into Top10 competent cells </li> |
| − | - | + | <li> -made plasmid mini-preps from these transformed cells </li> </ul> |
| − | + | -characterise the plasmid stocks obtained (Nano Drop and agarose gel)<br> | |
| − | + | -characterised the genotype of wild-type (W3110) and mutant (JC28) strains:<br> | |
| − | + | <ul> | |
| − | + | <li> -genomic DNA extractions have been performed </li> | |
| + | <li> - <i> entC </i> gene has been amplified- PCR </li> | ||
| + | <li>-PCR products have been analysed- agarose gel </li> | ||
| + | </ul> | ||
| + | </p> | ||
<h3> Constitutive hemerythrin expression </h3> | <h3> Constitutive hemerythrin expression </h3> | ||
<h3> Overexpression of hemerythrin </h3> | <h3> Overexpression of hemerythrin </h3> | ||
Revision as of 21:00, 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 for further cloning experiments:
- -transformed the pSB1C3, pUC18 and pBSKII plasmids into Top10 competent cells
- -made plasmid mini-preps from these transformed cells
-characterised the genotype of wild-type (W3110) and mutant (JC28) strains:
- -genomic DNA extractions have been performed
- - entC gene has been amplified- PCR
- -PCR products have been analysed- agarose gel
