Griffitts:DNA sequencing

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
  • 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