Sequencing


	DNA Sequencing PE-ABI Big Dye Terminator Reactions

Below is the protocol used for DNA sequencing at the University of Delaware DNA sequencing core facility. It also indicates suggested amounts of DNA to submit for sequencing.

To download a program to read ABI sequence files:
Click Here for PC - this will directly download a program.
Click Here for MAC - this will link you to the PE site. Download Editview.

Reaction Set Up

Thermocycler Settings

Post Reaction Clean Up


	Recommended Quantities of DNA:

Single Stranded DNA: 50-100 ng

Double Stranded DNA: 200-500 ng

PCR product: 30-90 ng


	Sequencing reactions for the PE-ABI 377


	Reaction Mixtures: Prepare in thin walled micro tubes


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Reaction Ready Mix	4 mL
Template	Max of 5 mL
Primer	1.6 pmol
Deionized Water	to 10 mL


	Cycle Sequencing Using ABI Thermocycler 480

96^oC	30 seconds
50^oC	15 seconds
60^oC	4 minutes
25 Cycles


	Cycle Sequencing Using ABI Thermocycler 9600/9700

96^oC	10 seconds
50^oC	5 seconds
60^oC	4 minutes
25 Cycles


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	Post Reaction Clean Up:

Ethanol Precipitation: Transfer reaction mixture to 1.5 ml tube. Add 15 ml H2O 64 ml Ethanol Room temperature: 15 minutes. Spin tubes at maximum speed: 10 minutes. Remove supernatant. Pellet should NOT be visible Wash with 100 ml 70% Ethanol (vortex). Spin at maximum: 10 minutes. Remove supernatant Dry samples: Vacuum centrifuge-10 min Or heat to 90^oC for 1 minute in open tube.

		Samples are best stored dry at 4^oC


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	Resuspend in 6 ml of loading buffer Loading Buffer: 100 ml Deionized Formamide 20 ml 25mM EDTA (pH = 8.0) + Blue Dextran (50 mg/ml) Load 1 ml on 4.25% Sequencing Gel.

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This page was adapted from the ABI-PE Instruction manual. Link to *ABI-PE Homepage*