Exeter/1 August 2013

From 2013.igem.org

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=== Miniprep ===  
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{{:Team:Exeter/Template/Header}}
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B0015
 
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K592018
 
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S05058
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Please not that <i>EcoRI, XbaI, PstI</i> and <i>SpeI</i> are refereed to as "E, X, P and S" respectively throughout this page.
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=== Miniprep === __NOTOC__
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[http://parts.igem.org/Part:BBa_B0015 B0015]
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[http://parts.igem.org/Part:BBa_K592018 K592018]
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[http://parts.igem.org/Part:BBa_S05058 S05058]
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=== Group 2 ===
=== Group 2 ===
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Eluted with RNAse-free water (But possibly contaminated)! Digestion by RAchel and Flick using Victoria's recipe.
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Eluted with RNAse-free water (But possibly contaminated)! Digestion by Rachel and Flick using Victoria's recipe.
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Neither worked! No DNA visible.
Neither worked! No DNA visible.
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Take me back to the [https://2013.igem.org/Team:Exeter/Notebook notebook].
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  </div>
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{{:Team:Exeter/Template/Footer}}

Latest revision as of 19:35, 2 October 2013

Exeter iGEM 2013 · Paint by Coli



Please not that EcoRI, XbaI, PstI and SpeI are refereed to as "E, X, P and S" respectively throughout this page.

Miniprep

[http://parts.igem.org/Part:BBa_B0015 B0015]

[http://parts.igem.org/Part:BBa_K592018 K592018]

[http://parts.igem.org/Part:BBa_S05058 S05058]


Nanodrop

Culture Nanodrop concentration (ng/ul) for digest (ul)
B0015 30.9 8.1
K592018 106.2 2.4
S05058 58.2 4.3
RFP - -


Nanodrop of digests

We are worried that there is insufficient DNA in our restriction digests, as our gels have no bands.


Group 1

Eluted with purite water, digestion by Adam using Victoria's protocol.


1 - RBS + Cph8 (E+S) - 43.9 ng/ul

2 - B0015 (x+p) - 26.7 ng/ul

3 - Negative control - 23.3 ng/ul (gives a reading due to NEB2 and BSA)

4 - Positive control (E+S) - 37.3

5 - Positive control (X+P) - 18.8


Group 2

Eluted with RNAse-free water (But possibly contaminated)! Digestion by Rachel and Flick using Victoria's recipe.


A - RBS + Cph8 (E+X) - 89.3

B - RBS + cyan (X+E) - 63.5

C - B0015 (E+S) - 66.0

D - AMP plasmid (E+P+D) - 40.4

E - Positive control (E+S) - 51.5

F - Positive control (X+P) - 57.3

G - Negative control (E+S) - 33.9 (gives a reading due to NEB2 and BSA)

Third retry of digestion

We have new no-nuclease water from Paul.

Instead of using an RFP plasmid from a transformation/mini-prep, we're using one resuspended from a kit plate.


The reason we're not seeing anything on the gels may be because we're not adding enough DNA.

We've split into 2 teams : Clio and Victoria Recipes:


Victoria's Recipe

Culture no-nucleotide water DNA NEB2 BSA E X S P
1. RBS + Cph8 5.0 10 2.5 0.5 1.0 1.0 - -
2. RBS + cyan 5.0 10 2,5 0.5 1.0 1.0 - -
3. B0015 5.0 10 2.5 0.5 1.0 - 1.0 -
5. Positive control (E+S) 5.0 7 3 - 1.0 - 1.0 -
7. Negative control 15.0 - 3 - 1.0 - 1.0 -

37°C for 10mins, 80°C for 20mins.


Clio's Recipe

Culture water DNA 10x fast buffer w/green E X S P D
A. RBS + Cph8 3 10 5 1.0 1.0 - - -
B. RBS + cyan 3 10 5 1.0 1.0 - - -
C. B0015 3 10 5 1.0 - 1.0 - -
D. AMP plasmid 3 7 5 1.0 - - 1.0 1.0
E. Positive control RFP (X+P) 5 10 5 - 1.0 - 1.0 -
G. Negative control 13 - 5 1.0 - 1.0 - -

37°C for 30mins , 80°C for 20mins.


We didnt have much resuspended RFP (hence 7ul DNA used) so 1 each. We also didt have enough AMP plasmid to do one each.


Gel

Lane 1 - 1kb GeneRuler ladder

Lane 2 - 1. RBS + Cph8

Lane 3 - 2. RBS + Cyan

Lane 4 - 3. B0015

Lane 5 - 5. RFP control E+S

Lane 6 - 7. negative control

Lane 7 - A. RBS + Cph8

Lane 8 - B. RBS + cyan

Lane 9 - C. B0015

Lane 10 - D. AMP plasmid

Lane 11 - E. RFP control X+P

Lane 12 - G. Negative control

Lane 13 - 100bp plus DNA ladder.


Neither worked! No DNA visible.

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli
Retrieved from "http://2013.igem.org/Exeter/1_August_2013"