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Team:TU Delft/15 July 2010 content
From 2010.igem.org
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|1.5 μg isolated control plasmid pSB3T1 | |1.5 μg isolated control plasmid pSB3T1 | ||
|Buffer 2 | |Buffer 2 | ||
| - | |3 fragments | + | |3 fragments (1828, 1475, 222 bp) |
|- | |- | ||
|2 | |2 | ||
|1 μg isolated plasmid with OprG (culture 6) | |1 μg isolated plasmid with OprG (culture 6) | ||
|Buffer 2 | |Buffer 2 | ||
| - | |2 fragments | + | |2 fragments (1972, 1176 bp) |
|- | |- | ||
|3 | |3 | ||
|1 μg isolated plasmid with OprG (culture 7) | |1 μg isolated plasmid with OprG (culture 7) | ||
|Buffer 2 | |Buffer 2 | ||
| - | |1 fragments | + | |1 fragments (1972, 1176 bp) |
|- | |- | ||
|4 | |4 | ||
|1 μg isolated plasmid with AlnA | |1 μg isolated plasmid with AlnA | ||
|Buffer 2 | |Buffer 2 | ||
| - | |1 fragment | + | |1 fragment (3511 bp) |
|} | |} | ||
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|3 | |3 | ||
|Digested control plasmid (10 μL + 1 μL loadingbuffer) | |Digested control plasmid (10 μL + 1 μL loadingbuffer) | ||
| - | | | + | |1972, 1176 bp |
|- | |- | ||
|4 | |4 | ||
| Line 62: | Line 62: | ||
|5 | |5 | ||
|Digested culture 6 OprG (10 μL + 2 μL loadingbuffer) | |Digested culture 6 OprG (10 μL + 2 μL loadingbuffer) | ||
| - | | | + | |1828, 1475, 222 bp |
|- | |- | ||
|6 | |6 | ||
|Digested culture 7 OprG (10 μL + 2 μL loadingbuffer) | |Digested culture 7 OprG (10 μL + 2 μL loadingbuffer) | ||
| - | | | + | |1828, 1475, 222 bp |
|- | |- | ||
|7 | |7 | ||
| Line 74: | Line 74: | ||
|8 | |8 | ||
|Digested culture 11 AlnA (10 μL + 2 μL loadingbuffer) | |Digested culture 11 AlnA (10 μL + 2 μL loadingbuffer) | ||
| - | | | + | |3511 bp |
|} | |} | ||
Revision as of 11:02, 19 July 2010
Lab work
Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing
Some of transformations of yesterday containing the different ligations gave colonies (1 a 2 per plate). We picked as many colonies as possible of every plate and performed a colony PCR to check which colonies contained the right insert. At the same time we grow them overnight in 5 mL LB medium containing the appropriate antibiotic.
Emulsifier
The results from the Colony PCR were not conclusive yesterday. That is why we decided to digest the plasmids isolated from the cultures once more.
The plasmids were digested with HindIII. This should yield 3 bands in the lane with the plasmid containing RFP (neg control), 1 band in the lane with AlnA and 2 bands in the lane with OprG on gel.
| # | Digestion reaction | Used Buffer | Needed fragment |
| 1 | 1.5 μg isolated control plasmid pSB3T1 | Buffer 2 | 3 fragments (1828, 1475, 222 bp) |
| 2 | 1 μg isolated plasmid with OprG (culture 6) | Buffer 2 | 2 fragments (1972, 1176 bp) |
| 3 | 1 μg isolated plasmid with OprG (culture 7) | Buffer 2 | 1 fragments (1972, 1176 bp) |
| 4 | 1 μg isolated plasmid with AlnA | Buffer 2 | 1 fragment (3511 bp) |
Lane description:
| # | Description | Expected Length (bp) |
| 1 | SmarLadder marker (5 μL) | |
| 2 | Undigested control plasmid (5 μL + 1 μL loadingbuffer) | |
| 3 | Digested control plasmid (10 μL + 1 μL loadingbuffer) | 1972, 1176 bp |
| 4 | Undigested culture 6 OprG (5 μL + 1 μL loadingbuffer) | |
| 5 | Digested culture 6 OprG (10 μL + 2 μL loadingbuffer) | 1828, 1475, 222 bp |
| 6 | Digested culture 7 OprG (10 μL + 2 μL loadingbuffer) | 1828, 1475, 222 bp |
| 7 | Undigested culture 11 AlnA (5 μL + 1 μL loadingbuffer) | |
| 8 | Digested culture 11 AlnA (10 μL + 2 μL loadingbuffer) | 3511 bp |
