Exeter/30 August 2013
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Results from yesterday's transformations
OmpF + RBS 100 ul equal amounts - 44 colonies
OmpF + RBS 100 ul equal moles - 8 colonies
OmpF + RBS 400 ul equal amounts - 107 colonies
OmpF + RBS 400 ul equal moles - 35 colonies
RFP control equal amounts - 15 colonies
RFP control equal moles - 49 colonies
Ligation
The ligation was carried out using two methods: equal volumes and equal moles. To calculate the equimolar volumes, we used the following data:
Part | Part Length (bp) |
---|---|
AMP plasmid | 2155 |
OmpF (R0084) | 108 |
RBS (B0034) | 12 |
RFP | 774 |
The Ompf, B0034, AMP plasmid, and RFP were from the earlier digestions. We pippetted the following volumes into four 0.6ml tubes:
New Part | A (OmpF + B0034, eq/vol) | B (RFP control, eq/vol) | C (OmpF + B0034, equimolar) | C(RFP Control, equimolar) | |
---|---|---|---|---|---|
Plasmid | 2 ul | 2 ul | 2 ul | 2 ul | |
Ompf | 2 ul | - | - | - | |
Ompf (10x dilution) | - | - | 1.0 ul | - | |
B0034 | 2 ul | - | - | - | |
B0034 (100x dilution) | - | - | 0.8 ul | - | |
RFP | - | 2 ul | - | - | |
RFP (10x diltion) | - | - | - | 0.74 ul | |
T4 ligase buffer | 1.0 ul | 1.0 ul | 1.0 ul | 1.0 ul | |
T4 DNA ligase | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul | |
Non-nuclease water | 2.5 ul | 4.5 ul | 4.7 ul | 5.8 ul |
We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.
The tubes were incubated at 16C for 30 minutes and then 80C for 20 minutes.