26/07/13

From 2013.igem.org

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(Restriction enzyme digest of the DNA samples)
 
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#purification of the limonene biobrick - BBa_24
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<div style="font-family:arial;padding:5px;border-radius:5px;border:5px solid #FF2800;">
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*Plasmid Volume  
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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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**1.1 - 40ul
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!align="center"|[[Team:Leicester|Home]]
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**1.2 - 40ul
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!align="center"|[[Team:Leicester/Team|Team]]
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**1.3 - 46ul
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!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile]
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**1.4 - 39ul
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!align="center"|[[Team:Leicester/Project|Project]]
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**1.5 - 41ul
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!align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]]
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**1.2 - 32ul
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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>
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{|border=1
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|Sample||Plasmid Volume(ul)||DNA concentration (ng/ul)
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|-
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|1.1||40||131.8
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|-
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|1.2||40||77.1
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|-
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|1.3||46||56.8
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|-
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|1.4||39||109
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|-
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|1.5||41||129.1
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|-
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|1.6||32||93.3
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|}
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*measured DNA concentration using nanodrop
*measured DNA concentration using nanodrop
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**DNA concentration (ng)
 
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**1.1 - 131.8
 
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**1.2 - 77.1
 
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**1.3 - 56.8
 
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**1.4 - 109
 
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**1.5 - 129.1
 
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**1.2 - 93.3
 
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*Restriction enzyme digest of the DNA samples
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==Restriction enzyme digest of the DNA samples==
**enzymes: XbaI, PstI
**enzymes: XbaI, PstI
**master mix:  
**master mix:  
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*To calculate it we divided 200ng by the concentration of each sample  
*To calculate it we divided 200ng by the concentration of each sample  
*The rest of the volume was made up with water to give a total of 5ul.
*The rest of the volume was made up with water to give a total of 5ul.
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*The samples were them incubated in a water bath at 37C by an hour
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*The samples were then incubated in a water bath at 37C for an hour
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*Agorose Gel preparation
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==Agorose Gel preparation==
***20ml of 5xTBE
***20ml of 5xTBE
***80ml of water
***80ml of water
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**Boil for 3 minutes, 1 minute at a time.
**Boil for 3 minutes, 1 minute at a time.
**Let it cool and add 5ul of ethidium bromide.
**Let it cool and add 5ul of ethidium bromide.
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**Pour and let set for 30 mins.
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**Pour and let it set for 30 mins.
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**Adding dye and and marker
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**Adding dye and marker
**Used orange g loading dye.
**Used orange g loading dye.
***2ul to each sample
***2ul to each sample

Latest revision as of 14:15, 22 August 2013

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


SamplePlasmid Volume(ul)DNA concentration (ng/ul)
1.140131.8
1.24077.1
1.34656.8
1.439109
1.541129.1
1.63293.3
  • measured DNA concentration using nanodrop

Restriction enzyme digest of the DNA samples

    • enzymes: XbaI, PstI
    • master mix:
      • Buffer - 14ul
      • H2O - 88.2ul
      • XbaI - 1.4ul
      • PstI - 1.4ul
      • Total volume - 105ul
  • Each sample contained 5ul of DNA and 15ul of the master mix.
  • The total amount of DNA in each sample was 200ng
  • To calculate it we divided 200ng by the concentration of each sample
  • The rest of the volume was made up with water to give a total of 5ul.
  • The samples were then incubated in a water bath at 37C for an hour

Agorose Gel preparation

      • 20ml of 5xTBE
      • 80ml of water
      • 0.8g of agarose
    • Boil for 3 minutes, 1 minute at a time.
    • Let it cool and add 5ul of ethidium bromide.
    • Pour and let it set for 30 mins.
    • Adding dye and marker
    • Used orange g loading dye.
      • 2ul to each sample
    • Used 1kb ladder, 5ul per 1 marker well.
  • Run the gel
  • Gel picture

IGEM lim restr dig 260713.jpg