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L A B O R A T O R Y

Part IV: DNA Labeling by Dideoxy Chain Termination

By following the directions under the tab above "Sequencing Service," you may submit amplified mt DNA samples for free sequencing at the DNA Learning Center. Follows are the DNA sequencing and DNA pruification protocols used.

Reagents	Equipment & Supplies	Shared Items
Sequencing master mix Mt control region amplicon	test tube, polypropylene 1-20 µl micropipet and tips 96-Well 0.2ml PCR Plate Sealing Foil	Thermal cycler ABI 3700 DNA Analyzer

Pre-lab Prep

Prepare a cycle sequencing master mix, with the following volumes. Mix by vortexing or pietting in and out.

	Per Reaction	10 Reactions
dH2O	4.5µl	45µl
Primer 1	1 µl	10 µl
Terminator Ready Reaction Mix	1µl	10 µl
5x Sequencing Buffer	1.5µl	15µl
Final Volume	8µl	80 µl

Procedure

Add 8 µl of master mix to each well on plate that will contain a sample.
Add 2 µl of mt amplicon (product) to each well containing 8 µl of master mix.
Cover plate with sealing foil.
Store 96-well plate on ice or in the freezer until ready to begin thermal cycling.
Program thermal cycler for 34 cycles according to the following cycle profile. The program may be linked to a 4°C cycle to hold samples after completing the cycle profile.

Hold 2 minutes - 98°C
Denaturing time and temperature 10 sec - 96°C
Annealing time and temperature 5 sec - 50°C
Extending time and temperature 4 min - 60°C
Hold (optional) - 4°C

M I T O C H ON D R I A L C O N T R O L R E G I O N

DNA Learning Center, Cold Spring Harbor Laboratory
Noncommercial, educational use only.
Kits available from Carolina Biological Supply Company.