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From 2013.igem.org

• purification of the limonene biobrick - BBa_24

1. Plasmid Volume

-1.1 - 40ul -1.2 - 40ul -1.3 - 46ul -1.4 - 39ul -1.5 - 41ul -1.2 - 32ul

1. 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

1. 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

1. 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.

1. Run the gel