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From 2010.igem.org

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 overnight 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 isolate the plasmid using Birnboim protocol.

The following plasmid concentrations were obtained:

BioBrick	Composed of	Concentration (ng/μL)
K398203	OprG-B0015 (transformant 6)
K398203	OprG-B0015 (transformant 7)
K398204	AlnA-B0015 (transformant 11)

The plasmids were subsequently digested with an enzyme that characteristic digest for all different plasmids. The plasmids were digested with HindIII. This should yield 3 bands in the lane with the plasmid pSB1T3 containing J04450 (neg control), 1 band in the lane with AlnA-B0015 and 2 bands in the lane with OprG-B0015 on gel.

#	Digestion reaction	Used Buffer
1	1.5 μg pSB1T3	Buffer 2 + BSA (BioLabs)
2	1 μg OprG-B0015 (transformant 6)	Buffer 2 + BSA (BioLabs)
3	1 μg OprG-B0015 (transformant 7)	Buffer 2 + BSA (BioLabs)
4	1 μg AlnA-B0015 (transformant 11)	Buffer 2 + BSA (BioLabs)

1% Agarose gel of digestion products:

Lane description:

#	Description	Expected Length (bp)
1	SmarLadder marker (5 μL)
2	Undigested pSB1T3 (5 μL + 1 μL loadingbuffer)
3	Digested pSB1T3 (10 μL + 1 μL loadingbuffer)	1972, 1176
4	Undigested OprG-B0015 (transformant 6) (5 μL + 1 μL loadingbuffer)
5	Digested OprG-B0015 (transformant 6) (10 μL + 2 μL loadingbuffer)	1828, 1475, 222
6	Digested OprG-B0015 (transformant 7) (10 μL + 2 μL loadingbuffer)	1828, 1475, 222
7	Undigested AlnA-B0015 (transformant 11) (5 μL + 1 μL loadingbuffer)
8	Digested AlnA-B0015 (transformant 11) (10 μL + 2 μL loadingbuffer)	3511