Sequencing PCR Reactions:

Cleaning/Quantifying

• Run 4 ul of your PCR reactions with 4ul of DNA low mass ladder (Life Technologies Cat.# 10068-013)
• Estimate the concentration of your PCR by comparing the brightness of the PCR bands to the ladder.
• Clean your PCRs using QIAquick Purification Kit (Quiagen Cat.# 28104 (50 samples) & 28106 (250 samples).
• Dry them down in the Speed Vac.
• Resuspend in water to obtain a concentration of 30-90ng/5ul.

Sequencing Reactions (10 ul reactions)

• ABI Prism Big Dye Cycle Sequencing Kit (Perkin Elmer Part # 4303152).
• Use a sequencing cocktail with the reaction Mix PE supplies diluted to 0.5X in a Dilution Buffer (200ul Tris 9.0, 5 ul 1M MgCl2, 795 ul H20).
• For each reaction, the cocktail contains 2 ul of the PE reaction Mix, 2ul of Dilution Buffer, and 1 ul of 10 uM Primer (5ul total).
• Add 5ul PCR product (at the appropriate concentration) to tubes containing 5ul of the cocktail.
• The program on the PE9700 is 94oC for 10 sec., 50oC for 5 sec., and 60oC for 4 min., linked to a 4oC soak.

Department of Biology, PO Box 400328 University of Virginia, Charlottesville, VA 22904-4328
Email: drt3b@virginia.edu  Phone:(434)982-5217