Griffitts:DNA sequencing

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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]

Brightness (compared to the

3 Kb band of the DNA ladder)

[DNA] cleaned up DNA ddH2O 1:10 primer Total
< 0.5X as bright < 6.25 ng/μL redo PCR redo PCR redo PCR redo PCR
0.5X as bright ~ 6.25 ng/μL 9.6 μL 0 μL 1.0 μL 10.6 μL
1X as bright ~ 12.5 ng/μL 4.8 μL 4.2 μL 1.0 μL 10.0 μL
2X brighter ~ 25 ng/μL 2.4 μL 6.6 μL 1.0 μL 10.0 μL
3X brighter ~ 37.5 ng/μL 1.6 μL 7.4 μL 1.0 μL 10.0 μL
4X brighter ~ 50 ng/μL 1.2 μL 7.8 μL 1.0 μL 10.0 μL
5X brighter ~ 62.5 ng/μL 1.0 μL 8.0 μL 1.0 μL 10.0 μL



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