Exeter/30 August 2013

From 2013.igem.org

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Revision as of 11:58, 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 digestions on the 29th. We pippetted the following volumes into four 0.6ml tubes:


New Part A (Cyan + B0015, eq/vol) B (RFP control, eq/vol) C (Cyan + B0015, equimolar) C(RFP Control, equimolar)
Plasmid 2 ul 2 ul 2 ul 2 ul
Cyan 2 ul - 0.66 -
B0015 2 ul - - -
B0015 (10x dilution) - - 1.2 ul -
RFP - 2 ul - 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..64 ul 5.76 ul


The tubes were incubated at 16C for 30 minutes and then 80C for 20 minutes.

Gel of Digestion

While carrying out the ligation, we ran a gel of our digested parts.

Lane Content
1 1kb Gene Ruler
2 Yellow (S05058)
3 Magenta
4 Yellow (K592010)
5 RFP
6 1kb Gene Ruler