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Sequencing prep

DNA Prep

  • Make a 1:10 dilution of each of your primers
    • 3 μL primer + 27 μL ddH20 will usually suffice
    • NOTE: Each sequence will require two samples—one with the upstream primer and one with the downstream primer
  • Label 1.5 mL microcentrifuge tubes
  • Add 1 μL of the appropriate primer to each tube
  • Add DNA from a  cleaned up   PCR  according to the table below
  • Add sterile ddH2O according to the table below
  • Report your sequencing reactions to Dr. Griffitts or  online
  • Take your sequencing reactions to 690 WIDB and put them in the refrigerator
    • NOTE: Make sure the liquid is at the bottom of the tube

Recipes

If you load 4 μL of our DNA ladder , the 3 Kb band will represent 12.5 ng/μL. To determine how much DNA to use for sequencing, compare the brightness (thickness) of the 3 Kb band to your DNA band then consult the table below. To sequence 600 to 800 bases, the BYU sequencing center requires 6.67 ng/μL. [1]