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From 2010.igem.org

Lab work

Characterization of Anderson RBS sequences

We tried to compensate for yesterdays delays. Thias performed a PCR reaction on short BioBrick inserts of J61100, J61101, J61107, J61117, J61127 and B0034. We wanted to investigate if this would save time in the long-run compared to transforming and plasmid isolation. The PCR reaction was run at varying annealing temperatures to figure out which temperature gave the best result.

After analysis on gel we deduced that a temperature of 50 °C was optimal.

At the end of the day we performed a Restriction digestion reaction on the amplified PCR products as well as the previously isolated I13401 and pSB3C5:

#	Sample	Enzyme 1	Enzyme 2	Buffer	BSA	Needed fragment
1	1.0 μg pSB1T3	EcoRI	PstI	H (Roche)	✗	‘X–linear pSB1T3–P’

In stead of 10 U/μL restriction enzyme we used 1 U/μL. Mixtures were incubated at 37 °C for 2.5 hours and stored at 4 °C overnight.