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 |
1-3 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.
