05/08/13

From 2013.igem.org

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{| style="color:#87EA00;background-color:#FFFFFF;" cellpadding="2" cellspacing="2" border="0" bordercolor="#000000" width="100%" align="center"
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!align="center"|[[Team:Leicester|Home]]
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!align="center"|[[Team:Leicester/Team|Team]]
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!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile]
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!align="center"|[[Team:Leicester/Project|Project]]
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!align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Leicester/Modeling|Modeling]]
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!align="center"|[[Team:Leicester/Notebook|Notebook]]
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!align="center"|[[Team:Leicester/Safety|Safety]]
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!align="center"|[[Team:Leicester/Attributions|Attributions]]
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|}
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==Isolation of ligated plasmid==
==Isolation of ligated plasmid==
*The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
*The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
* Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
* Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
* there were 6 tubes containing the plasmid.
* there were 6 tubes containing the plasmid.
-
* The plasmid concentration in each of the 6 tubes were measured.
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* The plasmid concentration in each of the 6 tubes were measured using nanodrop.
* 200ng of plasmid from each of the 6 different tube was used for digestion.
* 200ng of plasmid from each of the 6 different tube was used for digestion.
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{|border=1
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|sample||volume(ul)||concentration (ng/ul)||yield(ug)
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|-
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|5.1||99||75||7.4
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|-
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|5.2||100||44.2||4.4
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|-
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|5.3||98||46.7||4.6
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|-
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|10.1||46||61.8||2.84
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|-
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|10.2||44||77.1||3.39
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|-
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|10.3||47||13.7||0.64
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|}
   
   
==Digestion of ligated plasmid==
==Digestion of ligated plasmid==
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**1ul of PstI
**1ul of PstI
**varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
**varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
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** Final volume of each of the 6 tubes was 20ul.
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**Final volume of each of the 6 tubes was 20ul.
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==Gel Electrophoresis=
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==Gel Electrophoresis==
*The product of digestion was run on agarose gel
*The product of digestion was run on agarose gel
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** This was done in order to confirm the presence of the limonene biobrick
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**This was done in order to confirm the presence of the limonene biobrick
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[[File:iGem_Digested_050813.jpg]]
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*The lanes are in the following order:
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**Marker, Sample 5.1, Sample 5.2, Sample 5.3, Sample 10.1, Sample 10.2, Sample 10.3, Marker.
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*We were expecting to see a 2kb and 5kb band. These bands appeared for samples 5.1, 10.1,10.2 and 10.3. To further determine the composition of samples, we did a single digest on the samples.
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==Single digest==
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*For samples 5.1, 10.1, 10.2 and 10.3 two single digests each were made with EcoRI and PstI, giving a total of 8 samples.
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*The samples are run on 0.8% agarose gel.

Latest revision as of 12:30, 6 August 2013

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions


Contents

Isolation of ligated plasmid

  • The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
  • Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
  • there were 6 tubes containing the plasmid.
  • The plasmid concentration in each of the 6 tubes were measured using nanodrop.
  • 200ng of plasmid from each of the 6 different tube was used for digestion.
samplevolume(ul)concentration (ng/ul)yield(ug)
5.199757.4
5.210044.24.4
5.39846.74.6
10.14661.82.84
10.24477.13.39
10.34713.70.64

Digestion of ligated plasmid

  • Master Mix (changes were made from the protocol)
    • 2ul of NEB Buffer
    • 1ul of EcoRI
    • 1ul of PstI
    • varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
    • Final volume of each of the 6 tubes was 20ul.

Gel Electrophoresis

  • The product of digestion was run on agarose gel
    • This was done in order to confirm the presence of the limonene biobrick

IGem Digested 050813.jpg

  • The lanes are in the following order:
    • Marker, Sample 5.1, Sample 5.2, Sample 5.3, Sample 10.1, Sample 10.2, Sample 10.3, Marker.
  • We were expecting to see a 2kb and 5kb band. These bands appeared for samples 5.1, 10.1,10.2 and 10.3. To further determine the composition of samples, we did a single digest on the samples.

Single digest

  • For samples 5.1, 10.1, 10.2 and 10.3 two single digests each were made with EcoRI and PstI, giving a total of 8 samples.
  • The samples are run on 0.8% agarose gel.