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21/08/13
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==Reculturing Bacteria== | ==Reculturing Bacteria== | ||
| - | *The | + | *The ''P. putida'' grew overnight, which proved that we had viable cells. |
| + | *The bacteria was then re-streaked to single colonies and left to grow overnight in a 37C incubator. | ||
| + | ==Isolation of ''P. putida'' DNA == | ||
| + | *8 1.5ml samples were isolated from each of the 2 broths that were prepared yesterday. | ||
| + | *The first few steps of CTAB protocol was used to prep cells for DNA isolation; | ||
| + | *The 8 samples were spun down at 10000 rpm for 5 mins. | ||
| + | *The supernatant was discarded. | ||
| + | *cells were then resuspended in 1ml of TE. | ||
| + | *740ul of this new mixture was transferred to 8 new eppendorf tubes. | ||
| + | *20ul of lysozyme was added to each of these 8 samples. | ||
| + | * A maxwell robot was then used to complete the DNA extraction process. | ||
| + | ==Hydrodynamic shearing of Herring Sperm DNA== | ||
| + | *A 20G syringe was used ~24 times to squirt the herring sperm DNA in order to make it less viscous. | ||
| + | *30G syringe was also used | ||
| + | *This method seems to work better than the sonification. | ||
Latest revision as of 13:55, 22 August 2013
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Reculturing Bacteria
- The P. putida grew overnight, which proved that we had viable cells.
- The bacteria was then re-streaked to single colonies and left to grow overnight in a 37C incubator.
Isolation of P. putida DNA
- 8 1.5ml samples were isolated from each of the 2 broths that were prepared yesterday.
- The first few steps of CTAB protocol was used to prep cells for DNA isolation;
- The 8 samples were spun down at 10000 rpm for 5 mins.
- The supernatant was discarded.
- cells were then resuspended in 1ml of TE.
- 740ul of this new mixture was transferred to 8 new eppendorf tubes.
- 20ul of lysozyme was added to each of these 8 samples.
- A maxwell robot was then used to complete the DNA extraction process.
Hydrodynamic shearing of Herring Sperm DNA
- A 20G syringe was used ~24 times to squirt the herring sperm DNA in order to make it less viscous.
- 30G syringe was also used
- This method seems to work better than the sonification.
