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SamplePlasmid Volume(ul)DNA concentration (ng/ul)
  • 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 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 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