Some of the content on this website requires JavaScript to be enabled in your web browser to function as intended. While the website is still usable without JavaScript, it should be enabled to enjoy the full interactive experience.

Skip to Main Content

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

Contact Us

Macromolecular Analysis Core
Sequencing Core
AgC 5024B
766-2922
Macro Core
Physical Sciences 108
766-3688
UW Campus
Phone: 307-766-2922
Email: uwmac@uwyo.edu
Macromolecular Analysis Core
Dept 3944
1000 E University Ave
Laramie, WY 82071
logopicTMtrans2.gif

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.

Share This Page:

Footer Navigation

University of Wyoming
 
1000 E. University Ave. Laramie, WY 82071 // UW Operators (307) 766-1121 // Contact Us