Exeter/24 July 2013

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(Created page with " == Minipreps using the Qiagen kit == We miniprepped: * CcaR * Promoter, RBS x 3 replicates * CcaS * B0034 * Magenta * OmpR promoter * OmpR * YF1 * Lambda inverta * F...")
 
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==Results of liquid cultures== __NOTOC__
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Some of our liquid cultures didn't work; the cells died in the antibiotic. Mildly frustrating. The failed cultures were:
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*OmpR (BBa_K098011)
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*Lambda inverter system (BBa_Q04510)
== Minipreps using the Qiagen kit  ==
== Minipreps using the Qiagen kit  ==
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== Glycerol stocks ==
== Glycerol stocks ==
-
We made glycerol stocks of what is above, then stored them at -80<sub>o</sub>C
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We made glycerol stocks of what is above, then stored them at -80<sup>o</sup>C
For future reference, to get glycerol stocks back as plates, take a loop and streak on gel.  Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.
For future reference, to get glycerol stocks back as plates, take a loop and streak on gel.  Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.
Line 37: Line 49:
We then made a gel for digests using ethidium bromide.
We then made a gel for digests using ethidium bromide.
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== NanoDrop from miniprep ==
== NanoDrop from miniprep ==
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{| class="wikitable"
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|-
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! Part !! Concentration (ng/&micro;l)
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|-
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| CcaR ||324.6
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|-
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| CcaS || 264.4
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|-
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| Yellow || 64.3
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|-
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| Magenta || 102.7
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|-
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| RBS || 48.9
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|-
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| Promoter, RBS, #1 || 21.5
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|-
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| Promoter, RBS, #2 || 22.6
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|-
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| Promoter, RBS, #3 || 22.0
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|-
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| Fix L || 31.9
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|-
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| Fix J || 35.4
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|-
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| YF1 || 27.1
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|-
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| ''ompC'' || 28.2
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|}
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No SureClean was needed.
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==Digestion==
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We then cut the genes out of the plasmid using <i>Xbal</i> and <i>Pstl</i>.  Prepare to run on a gel.
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Each eppendorf contains:
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* 12 &micro;l purite water
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* 2 &micro;l 10X FastDigest Buffer w.Green
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* 0.5 &micro;l Xbal
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* 0.5 &micro;l Pstl
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* 5 &micro;l DNA
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== In gel ==
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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 || Ladder (1kb Gene Ruler)
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|-
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| 2 || Promoter, RBS, #1
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|-
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| 3 || B0034
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|-
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| 4 || CcaS
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|-
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| 5 || Fix J promoter (a.k.a Fix L)
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|-
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| 6 || Yellow
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|-
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| 7 || Promoter, RBS, #2
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|-
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| 8 || Fix J
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|-
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| 9 || OmpR promoter (a.k.a ''ompC'')
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|-
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| 10 || Promoter, RBS, #3
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|-
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| 11 || Magenta
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|-
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| 12 || CcaR
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|-
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| 13 || YF1
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|-
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| 14 || Ladder (1kb Gene Ruler)
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|}
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[[Image:24th_july_gel.jpg|center||500px|Image: 500 pixels]]
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The gel shows that some of our digestions appear to have worked (our yellow pigment in lane 6 has a clear band at about ~700bp, where would expect) but others appear not to have worked, or have bands that don't match our expected genes.
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==Cph8 gel==
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We're still uncertain about the presence of a <i>PstI</i> cut site in BBa_K322124 (cph8, our red light sensor), so we want to try cutting it with different combination of <i>EcoRI</i>, <i>XbaI</i>, <i>SpeI</i> and <i>PstI</i>. We have five replicates of cph8 plasmid from previous MiniPreps.
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* cph8 #3
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* cph8 #4
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* cph8 #5
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* cph8 #6
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* cph8 #7
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Unfortunately, they were only cut with <i>Xbal</i> and <i>Pstl</i> (Fran...), but this gel can be correctly run tomorrow with relative ease.
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The lanes were run in the order above, flanked by a 1kb Gene Ruler.
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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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</div>
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{{:Team:Exeter/Template/Footer}}

Latest revision as of 20:24, 2 October 2013

Exeter iGEM 2013 · Paint by Coli

Results of liquid cultures

Some of our liquid cultures didn't work; the cells died in the antibiotic. Mildly frustrating. The failed cultures were:

  • OmpR (BBa_K098011)
  • Lambda inverter system (BBa_Q04510)

Minipreps using the Qiagen kit

We miniprepped:

  • CcaR
  • Promoter, RBS x 3 replicates
  • CcaS
  • B0034
  • Magenta
  • OmpR promoter
  • OmpR
  • YF1
  • Lambda inverta
  • Fix J
  • Yellow
  • Fix J promoter


Glycerol stocks

We made glycerol stocks of what is above, then stored them at -80oC

For future reference, to get glycerol stocks back as plates, take a loop and streak on gel. Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.


We then made a gel for digests using ethidium bromide.

NanoDrop from miniprep

Part Concentration (ng/µl)
CcaR 324.6
CcaS 264.4
Yellow 64.3
Magenta 102.7
RBS 48.9
Promoter, RBS, #1 21.5
Promoter, RBS, #2 22.6
Promoter, RBS, #3 22.0
Fix L 31.9
Fix J 35.4
YF1 27.1
ompC 28.2


No SureClean was needed.

Digestion

We then cut the genes out of the plasmid using Xbal and Pstl. Prepare to run on a gel.

Each eppendorf contains:

  • 12 µl purite water
  • 2 µl 10X FastDigest Buffer w.Green
  • 0.5 µl Xbal
  • 0.5 µl Pstl
  • 5 µl DNA

In gel

Lane Content
1 Ladder (1kb Gene Ruler)
2 Promoter, RBS, #1
3 B0034
4 CcaS
5 Fix J promoter (a.k.a Fix L)
6 Yellow
7 Promoter, RBS, #2
8 Fix J
9 OmpR promoter (a.k.a ompC)
10 Promoter, RBS, #3
11 Magenta
12 CcaR
13 YF1
14 Ladder (1kb Gene Ruler)
Image: 500 pixels

The gel shows that some of our digestions appear to have worked (our yellow pigment in lane 6 has a clear band at about ~700bp, where would expect) but others appear not to have worked, or have bands that don't match our expected genes.

Cph8 gel

We're still uncertain about the presence of a PstI cut site in BBa_K322124 (cph8, our red light sensor), so we want to try cutting it with different combination of EcoRI, XbaI, SpeI and PstI. We have five replicates of cph8 plasmid from previous MiniPreps.

  • cph8 #3
  • cph8 #4
  • cph8 #5
  • cph8 #6
  • cph8 #7

Unfortunately, they were only cut with Xbal and Pstl (Fran...), but this gel can be correctly run tomorrow with relative ease.

The lanes were run in the order above, flanked by a 1kb Gene Ruler.

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli
Retrieved from "http://2013.igem.org/Exeter/24_July_2013"