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From 2013.igem.org

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