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Core DNA Sequencing Protocols|University of Wyoming | Macromolecular Analysis Core

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Macromolecular Analysis Core
Physical Sciences 108
UW Campus
Macromolecular Analysis Core
Dept 3944
1000 E University Ave
Laramie, WY 82071

Most of this is taken from:

BigDye® Terminator v3.1 Cycle Sequencing Kit Protocol

This publication is available from Applied Biosystems as a pdf document.  Click here for a copy in pdf format.

Cycle Sequencing Single- and Double-Stranded DNA

Using BigDye® Terminator v1.1/3.1 Sequencing Buffer

The BigDye® Terminator v1.1/3.1 Sequencing Buffer (5X)* is supplied at a 5X concentration. If you use it for sequencing reactions, be sure the final reaction volume is at a concentration of 1X. For example, for a half reaction in 20 µL final volume, you would use 4 µL of ready reaction premix and 2 µL of BigDye sequencing buffer
as shown below.




My typical reaction

My reaction for some plasmids

Ready Reaction Premix


4 µl

2 µl

2 µl

BigDye Sequencing Buffer


2 µl

1 µl


3.2 pmol

3.2-5 pmol

3.2-5 pmol


See below

See below

See below


to 20 µl

to 10 µl

to 5 µl

Final Volume


20 µl

10 µl

5 µl

Note: The use of this buffer without optimization may result in deterioration of sequence quality. Applied Biosystems does not support diluted reactions or guarantee the performance of BigDye® chemistry when it is diluted.

*The BigDye Terminator v1.1/3.1 Sequencing Buffer is intended for use only with BigDye Terminator v1.1/3.l Cycle Sequencing Kits.

DNA Quantity – Applied Biosystems recommendations:

Quantitating DNA - If possible, quantitate the amount of purified DNA by measuring the absorbance at 260 nm or by some other method.  We have a NanoDrop spectrophotometer available in the Teaching/Prep Lab for this purpose.

Template Quantity
The table below shows the amount of template to use in a cycle sequencing reaction.



PCR product:

100-200 bp
200-500 bp
500-1000 bp
1000-2000 bp
>2000 bp


1-3 ng
3-10 ng
5-20 ng
10-40 ng
20-50 ng


25-50 ng


150-300 ng

Cosmid, BAC

0.5-1.0 µg

Bacterial genomic DNA

2-3 µg

Note: In general, higher DNA quantities give higher signal intensities.

The template quantities stated above should work with all primers.
The amount of PCR product to use in sequencing also depends on the length and purity of the PCR product.

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