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Team:Groningen/Template/MODULE/Notebook/secretion/week8
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| - | + | <b>goal:</b> obtain the anti <i>Staphylococcus aureus</i> (p2s) and the <i>Pseudomonas aeruginosa</i>(pLASs). | |
| + | |||
| + | A decision was made to assemble the P2s pLASs with gibson assembly. An RBS is downstream attached of <i>P2</i>, <i>dspB</i> and <i>pLAS</i> with tail PCR. | ||
| + | Afterwards a gibson tail was attached with pcr to <i>p2+rbs</i>, <i>dspB+rbs</i>, <i>aiiA</i> and double terminator. | ||
| + | Then 59ng of <i>p2</i>, 400ng of <i>ssUSPDspB45</i>, 65ng of <i>nisA</i> and 50ng of Double terminator and incubated for 1:30 hours at 50 degrees celcius. | ||
| + | The gibson fragment was amplified with PCR after 20 cycles. With help of gradient PCR, we found the exact temperature of annealing. The p2s was then cloned into pSB1C3 and sequenced accordingly. | ||
| + | The p2s system was verified through restriction analysis and sequencing. | ||
| + | For the pLASs 70ng of <i>pLAS</i>, 400ng of <i>dspB+RBS</i>, 290ng of <i>aiiA</i> and 50ng of double terminator. Though after amplifying the fragment and loading it on gel, only a 1200bp band could be found. | ||
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Revision as of 15:57, 17 October 2014
goal: obtain the anti Staphylococcus aureus (p2s) and the Pseudomonas aeruginosa(pLASs).
A decision was made to assemble the P2s pLASs with gibson assembly. An RBS is downstream attached of P2, dspB and pLAS with tail PCR.
Afterwards a gibson tail was attached with pcr to p2+rbs, dspB+rbs, aiiA and double terminator.
Then 59ng of p2, 400ng of ssUSPDspB45, 65ng of nisA and 50ng of Double terminator and incubated for 1:30 hours at 50 degrees celcius.
The gibson fragment was amplified with PCR after 20 cycles. With help of gradient PCR, we found the exact temperature of annealing. The p2s was then cloned into pSB1C3 and sequenced accordingly.
The p2s system was verified through restriction analysis and sequencing.
For the pLASs 70ng of pLAS, 400ng of dspB+RBS, 290ng of aiiA and 50ng of double terminator. Though after amplifying the fragment and loading it on gel, only a 1200bp band could be found.
