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DNA Cycle Sequencing Kit

Sequencing with fluorescently labeled primers

Product Cat. No. Amount Price (EUR) Buy / Note
DNA Cycle Sequencing Kit PCR-401S 100 reactions 150,00 Add to Basket/Quote Add to Notepad
DNA Cycle Sequencing Kit PCR-401L 500 reactions 600,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Related products:

  • Fluorescent-labeled Oligonucleotides
  • Custom Oligonucleotides
  • Standard Primers
  • Sequencing Pol
  • Dideoxynucleotides (ddNTPs)
  • Sequencing Service

Description:
DNA Cycle Sequencing Kit is designed for DNA sequencing based on the Sanger Method (dideoxy chain termination method). It 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 specifically engineered Taq polymerase showing an equal capability of incorporating ddNTPs and dNTPs. This guarantees the generation of uniform and easy to read sequence band patterns at lowest background. A minimal band compression of GC-rich DNA regions is achieved by optimally balanced termination mixtures containing 7-deaza-dGTP.
The reaction chemistry of the kit is optimized for automated DNA sequencers and requires fluorescent-labeled primers.

Kit contents:
Terminator A (blue cap)
dNTP mix containing ddATP

Terminator C (blue cap)
dNTP mix containing ddCTP

Terminator G (blue cap)
dNTP mix containing ddGTP

Terminator T (blue cap)
dNTP mix containing ddTTP

Cycle sequencing polymerase (red cap)
4 units/μl

Cycle sequencing buffer (green cap)
10x conc.

Stop solution (purple cap)
95 % formamide containing EDTA, bromophenol blue, and xylene cyanol FF

PCR-grade water (white cap)

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.

Labeled Primers:
The kit is optimized for cycle sequencing using fluorescent-labeled 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-labeled primers to light.

Premix:
First prepare the following premix in a microcentrifuge tube:

4 μl10x Sequencing Buffergreen cap
1-2 pmolfluorescent-labeled Primer-
500-250 fmol or 30-150 ng/kbDNA-
1 μlSequencing Polred cap
fill up to 20 μlPCR-grade waterwhite cap

Mix by pipetting up and down several times.

Recommended assay preparation:
1) Transfer 4 μl of each Terminator A, C, G, and T (blue caps) into four separate and correspondingly marked tubes
2) Add 4 μl 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 °C2 min1x
denaturation95 °C30 sec20-30x
annealing60 °C30 sec20-30x
elongation72 °C60 sec20-30x

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

Selected References:
Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463.