22/08/13

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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</div>
==Measuring the concentrations of isolated ''P. putida'' DNA from the previous day==
==Measuring the concentrations of isolated ''P. putida'' DNA from the previous day==
*Measuring concentration using nanodrop
*Measuring concentration using nanodrop
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==Preparing samples to run on 1% gel==
==Preparing samples to run on 1% gel==
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*500ng of each of the isolated ''P. putida'' DNA sample is run on gel
 +
*Volumes are adjusted to 20ul per well accordingly (includes DNA, sterile H2O and OG)
 +
*5ul of orange G dye is added to each sample
 +
*5ul of 1kb ladders are used
 +
*For sheared DNA, dilutions were loaded
 +
**1ul of DNA was diluted in 9ul of 1xTE buffer to make up 10x dilution
 +
**1ul from 10x dilution was put in 9ul of 1xTE buffer to make up 100x dilution
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*Dilutions were nanodropped and the concentration for 100 fold dilution was 121 ng/ul
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*200ng of herring sperm DNA was run on gel
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*One lane was left for control, non-sheared DNA, also 100 fold dilution
==Running gel of ''P. putida'' DNA and Herring sperm DNA==
==Running gel of ''P. putida'' DNA and Herring sperm DNA==
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Samples in lanes goes as follows: 1 Kb ladder, 2, 4, 6, 8, 10, 12, 14, 16, 1 kb ladder, 100x dilution of sheared herring sperm DNA, control 100x diluted herring sperm DNA
Samples in lanes goes as follows: 1 Kb ladder, 2, 4, 6, 8, 10, 12, 14, 16, 1 kb ladder, 100x dilution of sheared herring sperm DNA, control 100x diluted herring sperm DNA
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==Running sonicated herring sperm DNA on 0.8% gel==
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*3 lanes for running:
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**Sheared and sonicated
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**Unsheared and sonicated
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**Unsheared and unsonicated
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*5ul of 1kb marker is used
 +
*The DNA samples are diluted 100 fold and 200ng of sample is run.
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*Volume of sterile water is adjusted appropriately to make up 20ul of sample per well, including 5ul of orange G dye
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[[File:igem_shear_son_220813.jpg]]
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*Not visible on the computer file, but picture shows a very slight decrease in sheared/sonificated and unsheared/sonicated samples vs the unsheared/unsonificated control
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*The difference is not significant, but the viscosity has changed when mechanically spreading the DNA on polystyrene, though it is not that evident on the gel.
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==Making up a sheared/sonicated dilution to leave on bench overnight==
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*500ul of sheared/sonicated DNA
 +
*500ul of 1xTE
 +
*Mix
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*Leave on bench overnight
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==Incubating sheared/sonicated dilution at 37C on a shaking rack==
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*10ul of 1xTE added to 2ul of sheared/sonicated DNA
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*Mix
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*Incubated overnight at 37C on a shaking rack

Latest revision as of 13:57, 23 August 2013

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

Contents

Measuring the concentrations of isolated P. putida DNA from the previous day

  • Measuring concentration using nanodrop
  • Blanking against buffer
Sample nr.Concentration ng/ul260/280260/230
2441.81.15
430.61.81.29
642.31.811.05
835.61.851.26
1038.21.841.28
1240.61.831.45
1439.91.881.47
1618.51.170.67

Preparing samples to run on 1% gel

  • 500ng of each of the isolated P. putida DNA sample is run on gel
  • Volumes are adjusted to 20ul per well accordingly (includes DNA, sterile H2O and OG)
  • 5ul of orange G dye is added to each sample
  • 5ul of 1kb ladders are used
  • For sheared DNA, dilutions were loaded
    • 1ul of DNA was diluted in 9ul of 1xTE buffer to make up 10x dilution
    • 1ul from 10x dilution was put in 9ul of 1xTE buffer to make up 100x dilution
  • Dilutions were nanodropped and the concentration for 100 fold dilution was 121 ng/ul
  • 200ng of herring sperm DNA was run on gel
  • One lane was left for control, non-sheared DNA, also 100 fold dilution

Running gel of P. putida DNA and Herring sperm DNA

Igem putida 220813.jpg


Samples in lanes goes as follows: 1 Kb ladder, 2, 4, 6, 8, 10, 12, 14, 16, 1 kb ladder, 100x dilution of sheared herring sperm DNA, control 100x diluted herring sperm DNA

Running sonicated herring sperm DNA on 0.8% gel

  • 3 lanes for running:
    • Sheared and sonicated
    • Unsheared and sonicated
    • Unsheared and unsonicated
  • 5ul of 1kb marker is used
  • The DNA samples are diluted 100 fold and 200ng of sample is run.
  • Volume of sterile water is adjusted appropriately to make up 20ul of sample per well, including 5ul of orange G dye

Igem shear son 220813.jpg

  • Not visible on the computer file, but picture shows a very slight decrease in sheared/sonificated and unsheared/sonicated samples vs the unsheared/unsonificated control
  • The difference is not significant, but the viscosity has changed when mechanically spreading the DNA on polystyrene, though it is not that evident on the gel.

Making up a sheared/sonicated dilution to leave on bench overnight

  • 500ul of sheared/sonicated DNA
  • 500ul of 1xTE
  • Mix
  • Leave on bench overnight

Incubating sheared/sonicated dilution at 37C on a shaking rack

  • 10ul of 1xTE added to 2ul of sheared/sonicated DNA
  • Mix
  • Incubated overnight at 37C on a shaking rack
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