Exeter/30 August 2013

From 2013.igem.org

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The Ompf, B0034, AMP plasmid, and RFP were from the earlier digestions.
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K592011, B0015, AMP plasmid, and RFP were from the earlier digestions.
We pippetted the following volumes into four 0.6ml tubes:
We pippetted the following volumes into four 0.6ml tubes:

Revision as of 11:34, 2 September 2013

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
K592011 702
RBS (B0015) 129
RFP 774


K592011, B0015, 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.