Applied Biosystems™

Thermo Sequenase Cycle Sequencing Kit

Catalog number:  785001KT

Related applications:

Sequencing

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Description

The Thermo Sequenase Cycle Sequencing Kit allows for equally efficient incorporation of both ddNTPs and dNTPs in cycle sequencing reactions resulting in very uniform band intensities which can be read accurately. The kit offers the choice of a three-dNTP extended primer method with internal α-labeled dNTPs or a 5' end-labeled primer method.

Obtain High Quality Sequence
Like Sequenase DNA Polymerase, Thermo Sequenase generates sequences with uniform band intensities and does not have the variability in band intensities of Taq DNA polymerase.

Flexible Labeling
The kit can be used for either internal labeling with α-labeled dNTPs or with 5' end-labeled primers.

Use Less Template
As little as 5 fmol of single-stranded or 20 fmol of double-stranded template is needed.

Read Through Secondary Structure
Regions with palindromes, hairpins or base repeats are more easily resolved with Thermo Sequenase and cycle sequencing methods.

Resolve Gel Compressions with 7-deaza-dGTP Nucleotide Mixes
When DNA is synthesized with 7-deaza-dGTP instead of dGTP, weaker secondary structures are formed, thus reducing compression artifacts in sequencing gels.

Eliminate Weak Bands with Pyrophosphatase
Occasional weak bands occur with prolonged reaction or cycle times. This is caused by sequence-specific pyrophosphorolysis which is catalyzed by the polymerase. The addition of pyrophosphatase minimizes pyrophosphorolysis and can prevent incorrect base-calling due to weak band intensities.

Important Notes:

The formulation of Thermo Sequenase DNA Polymerase in this kit requires the use of a glycerol tolerant DNA sequencing gel to eliminate glycerol-induced distortion of bands at approximately 350 to 600 bases beyond the primer. Information on use is included in the kit protocol. If this region is beyond your reading range, Tris-Borate-EDTA Buffer (TBE) may be used.

References:
TABOR, S. AND RICHARDSON, C. C. (1995) Proc. Natl. Acad. Sci. USA 92, p. 6339-6343.
SAMOLS, S. B., MCARDLE, B. F., RUAN, C. C., VANDERHORN, P. B. AND FULLER, C. W. (1995) Comments 22, No. 2, United States Biochemical Corp., Cleveland, OH.
RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, No.1, United States Biochemical Corp., Cleveland, OH.
TABOR, S. AND RICHARDSON, C. C. (1990) J. Biol. Chem. 265, 6447-6458.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

For Use With (Application): DNA Sequencing, Sanger Sequencing
Description: Thermo Sequenase Cycle Sequencing Kit, –20°C Stored
Includes: Thermo Sequenase with thermostable Pyrophosphatase, Control DNA pUC18, Control Primer (-40 Forward, 23-mer), Cycle Mix 7-deaza-dGTP, Cycle Mix dATP, Cycle Mix dTTP, Cycle Mix dCTP, ddGTP Termination Mix (7-deaza-dGTP), ddATP Termination Mix (7-deaza-dGTP), ddTTP Termination Mix (7-deaza-dGTP), ddCTP Termination Mix (7-deaza-dGTP), Reaction Buffer (concentrate), Stop Solution, Protocol Book
Product Type: Thermo Sequenase Cycle Sequencing Kit
Quantity: 1 Kit
Storage Requirements: –20°C

Contents & storage

Kit Components:
Thermo Sequenase with thermostable Pyrophosphatase
Control DNA pUC18
Control Primer (-40 Forward, 23-mer)

Cycle Mix 7-deaza-dGTP
Cycle Mix dATP
Cycle Mix dTTP
Cycle Mix dCTP
ddGTP Termination Mix (7-deaza-dGTP)
ddATP Termination Mix (7-deaza-dGTP)
ddTTP Termination Mix (7-deaza-dGTP)
ddCTP Termination Mix (7-deaza-dGTP)

Reaction Buffer (concentrate)
Stop Solution
Protocol Book

This kit and all the enclosed reagents should be stored frozen at -20°C (NOT in a frost-free freezer). Keep all reagents on ice when removed from storage for use.