26/07/13

From 2013.igem.org

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[[File:iGEM_lim_restr_dig_260713.jpg]]
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<div style="font-family:arial;padding:5px;border-radius:5px;border:5px solid #FF2800;">
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*purification of the limonene biobrick - BBa_24
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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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#Plasmid Volume
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!align="center"|[[Team:Leicester|Home]]
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-1.1 - 40ul
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!align="center"|[[Team:Leicester/Team|Team]]
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-1.2 - 40ul
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!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile]
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-1.3 - 46ul
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!align="center"|[[Team:Leicester/Project|Project]]
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-1.4 - 39ul
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!align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]]
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-1.5 - 41ul
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!align="center"|[[Team:Leicester/Modeling|Modeling]]
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-1.2 - 32ul
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!align="center"|[[Team:Leicester/Notebook|Notebook]]
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#measured DNA concentration using nanodrop
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!align="center"|[[Team:Leicester/Safety|Safety]]
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*DNA concentration (ng)
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!align="center"|[[Team:Leicester/Attributions|Attributions]]
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-1.1 - 131.8
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|}
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-1.2 - 77.1
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</div>
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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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*enzymes: XbaI, PstI
 
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*master mix:
 
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Buffer - 14ul
 
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H2O - 88.2ul
 
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XbaI - 1.4ul
 
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PstI - 1.4ul
 
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Total volume - 105ul
 
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Each sample contained 5ul of DNA and 15ul of the master mix.
 
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The total amount of DNA in each sample was 200ng
 
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To calculate it we divided 200ng by the concentration of each sample
 
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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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*Agorose Gel preparation
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{|border=1
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-20ml of 5xTBE
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|Sample||Plasmid Volume(ul)||DNA concentration (ng/ul)
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-80ml of water
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|-
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-0.8g of agarose
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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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|1.3||46||56.8
 +
|-
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|1.4||39||109
 +
|-
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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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Boil for 3 minutes, 1 minute at a time.
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*measured DNA concentration using nanodrop
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Let it cool and add 5ul of ethidium bromide.
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Pour and let set for 30 mins.
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*Adding dye and and marker
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==Restriction enzyme digest of the DNA samples==
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Used orange g loading dye.
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**enzymes: XbaI, PstI
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-2ul to each sample
+
**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
-
Used 1kb ladder, 5ul per 1 marker well.  
+
==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
*Run the gel
 +
*Gel picture
 +
[[File:iGEM_lim_restr_dig_260713.jpg]]

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

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