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.

Reagent
 Concentration
 Volume
 My typical reaction
 My reaction for some plasmids
Ready Reaction Premix
 2.5X
 4 µl
 2 µl
 2 µl
BigDye Sequencing Buffer
 5X
 2 µl
 1 µl
 —
Primer
 —
 3.2 pmol
 3.2-5 pmol
 3.2-5 pmol
Template
 —
 See below
 See below
 See below
Water
 —
 to 20 µl
 to 10 µl
 to 5 µl
Final Volume
 1X
 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.

Template
 Quantity
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
Single-stranded
 25-50 ng
Double-stranded
 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.