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Team:Groningen/Template/MODULE/Notebook/secretion/week9
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| + | After doing growing analyzing and miniprepping the correct clones, A1” and A3”was cut with EcoRI and SpeI. A2” and A4” was cut with XbaI and PstI and ligated afterwards | ||
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| + | <th>Name assembly</th><th>Upstream part</th><th>Downstream part</th><th>Backbone</th> | ||
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| + | <td>A1”’</td><td><i>pLAS</i>+RBS+<i>dspB</i></td><td>RBS+<i>aiiA</i>+Dterm</td><td>pSB1C3</td> | ||
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| + | <td>A2”’</td><td><i>pLAS</i>+RBS+<i>dspB+HIS</i>></td><td>RBS+<i>aiiA+HIS</i>+Dterm</td><td>pSB1C3</td> | ||
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Revision as of 21:08, 17 October 2014
September 29 - October 5
goal: Obtaining pLASs with 3-a assembly
Extra amounts of dspB, dspB+HIS, aiiA, aiiA+HIS,aiiA+HIS, pLAS, RBS, double terminator, pSB2k3, pSB1C3 and pSB1A3 were miniprepped to be used for the assembly
1 ug of aiiA, dspB, dspB+HIS and aiiA+HIS with XbaI and PstI and 1 ug of RBS was cut with EcoRI and SpeI
These were eventually ligated for 3 hours into pSB2K3 and transformed into E. coli NEB cells
| Name assembly | Upstream part | Downstream part | Backbone |
|---|---|---|---|
| A1’ | RBS | aiiA | pSB2K3 |
| A2’ | RBS | dspB | pSB2K3 |
| A3’ | RBS | dspB+HIS | pSB2K3 |
| A4’ | RBS | aiiA+HIS | pSB2K3 |
After growing them for 1 day, colony PCR was done on the correct clones, these were inoculated for plasmid extraction
Then A1’, A4’ was digested with XbaI and PstI. A2’ and A3’ was cut with EcoRI and SpeI. Then ligated
| Name assembly | Upstream part | Downstream part | Backbone |
|---|---|---|---|
| A1” | RBS + aiiA | Double terminator | pSB1A3 |
| A2” | pLAS | RBS + dspB | pSB1A3 |
| A3” | pLAS | RBS + dspB+HIS | pSB1A3 |
| A4” | RBS + aiiA+HIS | Double terminator | pSB1A3 |
After doing growing analyzing and miniprepping the correct clones, A1” and A3”was cut with EcoRI and SpeI. A2” and A4” was cut with XbaI and PstI and ligated afterwards
| Name assembly | Upstream part | Downstream part | Backbone |
|---|---|---|---|
| A1”’ | pLAS+RBS+dspB | RBS+aiiA+Dterm | pSB1C3 |
| A2”’ | pLAS+RBS+dspB+HIS> | RBS+aiiA+HIS+Dterm | pSB1C3 |
