Team:TU Delft/15 July 2010 content

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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.

1% Agarose gel of digestion products
# Digestion reaction Used Buffer
1 1.5 μg isolated control plasmid pSB3T1 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