Exeter/30 August 2013

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(Results from yesterday's transformations)
 
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== Results from yesterday's transformations ==
== Results from yesterday's transformations ==
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OmpF + RBS 100 ul equal amounts - 44 colonies
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<i>ompF</i> + RBS 100 &micro;l  equal amounts - 44 colonies
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OmpF + RBS 100 ul equal moles - 8 colonies
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<i>ompF</i> + RBS 100 &micro;l equal moles - 8 colonies
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OmpF + RBS 400 ul equal amounts - 107 colonies
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<i>ompF</i> + RBS 400 &micro;l equal amounts - 107 colonies
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OmpF + RBS 400 ul equal moles - 35 colonies
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<i>ompF</i> + RBS 400 &micro;l equal moles - 35 colonies
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RFP control equal amounts - 15 colonies
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[http://parts.igem.org/Part:BBa_J04450 RFP control] equal amounts - 15 colonies
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RFP control equal moles - 49 colonies
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[http://parts.igem.org/Part:BBa_J04450 RFP control] equal moles - 49 colonies
==Ligation==
==Ligation==
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K592011, B0015, AMP plasmid, and RFP were from the earlier digestions.
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K592011, B0015, AMP plasmid, and RFP were from the digestions on the 29th.
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We pippetted the following volumes into four 0.6ml tubes:
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We pipetted the following volumes into four 0.6ml tubes:
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| Plasmid || 2 ul || 2 ul || 2 ul || 2 ul  
| Plasmid || 2 ul || 2 ul || 2 ul || 2 ul  
|-
|-
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| Cyan || 2 ul || - || - || -
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| Cyan || 2 ul || - || 0.66 || -
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|-
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| Ompf (10x dilution) || - || - || 1.0 ul || -
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|-
|-
| B0015 || 2 ul || - || - || -
| B0015 || 2 ul || - || - || -
|-
|-
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| B0015 (100x dilution) || - || - || 0.8 ul || -
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| B0015 (10x dilution) || - || - || 1.2 ul || -
|-
|-
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| RFP || - || 2 ul || - || -
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| RFP || - || 2 ul || - || 0.74 ul
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|-
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| RFP (10x diltion) || - || - || - || 0.74 ul
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|-
|-
| T4 ligase buffer || 1.0 ul || 1.0 ul || 1.0 ul || 1.0 ul
| T4 ligase buffer || 1.0 ul || 1.0 ul || 1.0 ul || 1.0 ul
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| T4 DNA ligase || 0.5 ul || 0.5 ul || 0.5 ul || 0.5 ul  
| T4 DNA ligase || 0.5 ul || 0.5 ul || 0.5 ul || 0.5 ul  
|-
|-
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| Non-nuclease water ||  2.5 ul || 4.5 ul || 4.7 ul || 5.8 ul ||
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| Non-nuclease water ||  2.5 ul || 4.5 ul || 4..64 ul || 5.76 ul ||
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|}
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The tubes were incubated at 16<sup>o</sup>C for 30 minutes and then 80<sup>o</sup>C for 20 minutes.
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==Gel of Digestion==
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While carrying out the ligation, we ran a gel of our digested parts.
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{| class="wikitable"
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|-
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!Lane !! Content
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|-
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| 1 || 1kb Gene Ruler
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|-
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| 2 || K592011
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|-
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| 3 || B0015
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|-
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| 4 || AMP Plasmid
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|-
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| 5 || RFP
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|-
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| 6 || 1kb Gene Ruler
|}
|}
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We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.  
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Take me back to the [https://2013.igem.org/Team:Exeter/Notebook notebook].
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The tubes were incubated at 16C for 30 minutes and then 80C for 20 minutes.
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Latest revision as of 20:39, 2 October 2013

Exeter iGEM 2013 · Paint by Coli

Results from yesterday's transformations

ompF + RBS 100 µl equal amounts - 44 colonies

ompF + RBS 100 µl equal moles - 8 colonies

ompF + RBS 400 µl equal amounts - 107 colonies

ompF + RBS 400 µl 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 pipetted 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 16oC for 30 minutes and then 80oC 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 K592011
3 B0015
4 AMP Plasmid
5 RFP
6 1kb Gene Ruler

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli