Team:TU Delft/15 July 2010 content
From 2010.igem.org
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Revision as of 19:36, 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 |
1 | 1.5 μg isolated control plasmid pSB1T3 | Buffer 2 + BSA (BioLabs) |
2 | 1 μg isolated plasmid with OprG (culture 6) | Buffer 2 + BSA (BioLabs) |
3 | 1 μg isolated plasmid with OprG (culture 7) | Buffer 2 + BSA (BioLabs) |
4 | 1 μg isolated plasmid with AlnA | Buffer 2 + BSA (BioLabs) |
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 |
4 | Undigested culture 6 OprG (5 μL + 1 μL loadingbuffer) | |
5 | Digested culture 6 OprG (10 μL + 2 μL loadingbuffer) | 1828, 1475, 222 |
6 | Digested culture 7 OprG (10 μL + 2 μL loadingbuffer) | 1828, 1475, 222 |
7 | Undigested culture 11 AlnA (5 μL + 1 μL loadingbuffer) | |
8 | Digested culture 11 AlnA (10 μL + 2 μL loadingbuffer) | 3511 |