Line 282: | Line 282: | ||
-Decided an experimental outline <br> | -Decided an experimental outline <br> | ||
-In silico design of hemerythrin constructs <br> | -In silico design of hemerythrin constructs <br> | ||
− | -Synthetic genes and primers required for cloning have been ordered <br> | + | -Synthetic genes and primers required for cloning have been ordered <br> </p> |
<p> <b> <u> Week 2:</u> </b> <br> | <p> <b> <u> Week 2:</u> </b> <br> | ||
Line 289: | Line 289: | ||
-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) < | + | -Several experiments have been performed in order to monitor the growth of the wild type (W3110) and siderophore-deficient mutant (JC28) </p> |
<p> <b> <u> Week 3:</u> </b> <br> | <p> <b> <u> Week 3:</u> </b> <br> | ||
− | - | + | -Ensured we had sufficient amounts of plasmid for further cloning experiments: <br> |
<ul> | <ul> | ||
− | <li> | + | <li> transformed the pSB1C3, pUC18 and pBSKII plasmids into Top10 competent cells </li> |
− | <li> | + | <li> made plasmid mini-preps from these transformed cells </li> </ul> </p> |
− | - | + | <p> -Characterised the plasmid stocks obtained (Nano Drop and agarose gel)<br> |
− | - | + | -Characterised the genotype of wild-type (W3110) and mutant (JC28) strains:<br> |
<ul> | <ul> | ||
<li> -genomic DNA extractions have been performed </li> | <li> -genomic DNA extractions have been performed </li> |
Revision as of 21:02, 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 plasmid stocks obtained (Nano Drop and agarose gel)
-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