DNA Cycle Sequencing Kit - Sequencing with fluorescent labelled primers by Sanger Method. ! THIS PRODUCT IS DISCONTINUED !

Cycle Sequencing of cDNA - for Geneom Research ! DISCONTINUED !

Description:

The Maximo-DNA Cycle Sequencing Kit provides a powerful tool to derive rapidly DNA and gene sequence information as required in a multitude of molecular biological and biotechnological applications.

 

The performance of the kit is based on a enhanced Taq polymerase showing an equal capability of incorporating ddNTPs and dNTPs. As a result the Maximo-Cycle Sequencing-Kit offers uniform and easy to read sequence band patterns at lowest background.

An absolutely minimal band compression of GC-rich DNA regions is realized by optimally balanced termination mixtures containing 7-deaza-dGTP.

 

The reaction chemistry of the kit is optimized for automated DNA sequencers and requires labelled primers with fuorescent dyes.

Content:

Terminate solution A (blue cap): dNTP mix containing ddATP

Terminate solution C (blue cap): dNTP mix containing ddCTP

Terminate solution G (blue cap): dNTP mix containing ddGTP

Terminate solution T (blue cap): dNTP mix containing ddTTP

 

Cycle sequencing Polymerase (red cap): 4 Units/µl

 

Cycle sequencing Buffer (green cap): 10 fold

 

PCR-grade water (white cap)

 

Stopsolution (purple cap): 95 % formamide containing EDTA, bromophenol blue, and xylene cyanolFF

 

Shipping: shipped on blue ice

 

Storage Conditions: store at -20°C

 

Note: avoid multiple freeze / thaw cycles

 

Shelf Life: 18 months

 

References / Protocols / Notes / Recomendations / Tests

Cycle sequencing:
DNA cycle sequencing is a core technique in molecular biology allowing analysis of fmol-quantities DNA template. The enzymatic dideoxy chain termination method of Sanger relies on the linear amplification of a single-stranded template DNA using a single primer and thermostable polymerase. The synthesis of the complementary DNA strand starts at the specific priming site and ends with the incorporation of a chain-terminating dideoxynucleotide triphosphate (ddNTP). This generates a multitude of fragments terminated within the desired length of the sequence. By using the four different ddNTPs in four separate reaction vials, a set of extended primer strands terminated at each A, C, G, and T are obtained. When these fragments are separated on a suitable gel matrix the sequence information can be read from the order of the bands.

 

Labelled Primers:
The kit is optimized for cycle sequencing using fluorescent-labelled primers. The required 5’-end fluorescent label of the primer depends on the optical set-up of the used sequencing machine. Primers should typically be 20-25 nucleotides in length with a content of 50-60 % G+C. They should be checked to avoid forming of internal duplexes or mispriming to other sites of the template. Minimize the exposure of fluorenscent- labelled primers to light.

 

Stability tests / Quality control / Comparison

produced in ISO-certificated company

Premix:

Prepare the following premix in a microcentrifuge tube

Amount Component Codeing
 4 µl 10x Sequencing Buffer green cap
1-2 pmol fluorescent labelled Primer -

500-250 fmol

or 30-150 ng/kb

DNA -
1 µl Sequencing Polymerase red cap
fill up to 20 µl PCR-grade water white cap

Mix by pipetting up and down several times.

Recommended assay preparation:

1) Transfer 4µl of each Terminator A, C, G and T (blue caps) into four separate and correspondingly marked tubes

2) Add 4µl of the Premix to each tube and mix gently

 

Recommended cycling conditions:

Place the tubes in the thermal cycler and start the cycling program. The following parameters are recommended:

 

 Initial denaturation  95°C 2 min   1x
 denaturation 95°C   30 sec  20-30x
 annealing  60°C  30 sec  20-30x
 elongation 72°C    60 sec   20-30x

 

The annealing temperature depends on the primers used and should be 5-10 °C lower than its melting temperature. The melting temperature can be calculated for primers of up to 25 nucleotides using the formula: Tm=2(A+T)+4(G+C) A,T,G,C-number of respective nucleotides for optimal results an empirical optimization of the recommended parameters may be necessary for each new primer/template combination.

 

Analyzing the samples:
1) After cycling add 4 µl Stop Solution (purple cap) to each of the vials and mix again
2) If the samples cannot be analyzed immediately, they may be stored at-20°C for up to one week
3) Incubate the samples at 90°C for 2 min to denature the DNA
4) Load 3-5 µl of each reaction onto the gel

 

DNA Sequencing method by SANGER
DNA Sequencing Kit

 

 

 

 

 

 

Overview PCR Master Mixe:

 

BioStar 2 PCR mastermix with SYBRGreen blue dye, no rox

 

BioStar 3 PCR mastermix with SYBRGreen 60 nm LOW-ROX

 

BioStar 4 PCR mastermix with SYBRGreen 900 nm HIGH-ROX

BioStar 5 PCR mastermix

for probes

 

BioStar 6 PCR mastermix
for probes 60 mM LOW-ROX

 

BioStar 7 PCR mastermix
for probes 900 mM HIGH-ROX


Deutsche Beschreibung

 DNA Cycle Sequening Kit

Datasheet Cycle Sequening Kit

Cycle Sequencing with labeled primers
DNA cycle sequencing is a core technique in molecular biology allowing analysis of fmol-quantities DNA template. The enzymatic dideoxy chain termination method of Sanger relies on the linear amplification of a single-stranded template DNA using a single primer and thermostable polymerase
DNA Cycle Sequencing Kit.pdf
Adobe Acrobat Document 153.8 KB

Material Safety Datasheet


Matrix themes

Close