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Team:TU Delft/22 June 2010 content
From 2010.igem.org
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==Characterization of Anderson RBS sequences== | ==Characterization of Anderson RBS sequences== | ||
| - | We tried to compensate for yesterdays delays. Thias performed a [[Team:TU_Delft/protocols/ | + | We tried to compensate for yesterdays delays. Thias performed a [[Team:TU_Delft/protocols/colony_PCR|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. | After analysis on gel we deduced that a temperature of 50 °C was optimal. | ||
Latest revision as of 19:50, 5 August 2010
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.
