26/07/13

From 2013.igem.org

(Difference between revisions)
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[[File:iGEM_lim_restr_dig_260713.jpg]]
[[File:iGEM_lim_restr_dig_260713.jpg]]
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*purification of the limonene biobrick - BBa_24
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#purification of the limonene biobrick - BBa_24
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#Plasmid Volume  
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*Plasmid Volume  
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-1.1 - 40ul
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**1.1 - 40ul
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-1.2 - 40ul
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**1.2 - 40ul
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-1.3 - 46ul
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**1.3 - 46ul
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-1.4 - 39ul
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**1.4 - 39ul
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-1.5 - 41ul
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**1.5 - 41ul
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-1.2 - 32ul
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**1.2 - 32ul
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#measured DNA concentration using nanodrop
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*measured DNA concentration using nanodrop
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*DNA concentration (ng)
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**DNA concentration (ng)
**1.1 - 131.8
**1.1 - 131.8
**1.2 - 77.1
**1.2 - 77.1
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**1.2 - 93.3
**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
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*enzymes: XbaI, PstI
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**enzymes: XbaI, PstI
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*master mix:  
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**master mix:  
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**Buffer - 14ul
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***Buffer - 14ul
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**H2O - 88.2ul
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***H2O - 88.2ul
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**XbaI - 1.4ul
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***XbaI - 1.4ul
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**PstI - 1.4ul
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***PstI - 1.4ul
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**Total volume - 105ul
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***Total volume - 105ul
*Each sample contained 5ul of DNA and 15ul of the master mix.
*Each sample contained 5ul of DNA and 15ul of the master mix.
*The total amount of DNA in each sample was 200ng  
*The total amount of DNA in each sample was 200ng  
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*The samples were them incubated in a water bath at 37C by an hour
*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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*Agorose Gel preparation
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**20ml of 5xTBE
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***20ml of 5xTBE
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**80ml of water
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***80ml of water
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**0.8g of agarose
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***0.8g of agarose
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**Boil for 3 minutes, 1 minute at a time.
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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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**Used orange g loading dye.
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***2ul to each sample
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**Used 1kb ladder, 5ul per 1 marker well.
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*Boil for 3 minutes, 1 minute at a time.
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*Run the gel
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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
+
-
*Used orange g loading dye.
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**2ul to each sample
+
-
 
+
-
*Used 1kb ladder, 5ul per 1 marker well.
+
-
 
+
-
#Run the gel
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Revision as of 09:43, 29 July 2013

IGEM lim restr dig 260713.jpg

  1. purification of the limonene biobrick - BBa_24
  • Plasmid Volume
    • 1.1 - 40ul
    • 1.2 - 40ul
    • 1.3 - 46ul
    • 1.4 - 39ul
    • 1.5 - 41ul
    • 1.2 - 32ul
  • measured DNA concentration using nanodrop
    • DNA concentration (ng)
    • 1.1 - 131.8
    • 1.2 - 77.1
    • 1.3 - 56.8
    • 1.4 - 109
    • 1.5 - 129.1
    • 1.2 - 93.3
  • 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 them incubated in a water bath at 37C by 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 set for 30 mins.
    • Adding dye and and marker
    • Used orange g loading dye.
      • 2ul to each sample
    • Used 1kb ladder, 5ul per 1 marker well.
  • Run the gel
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