Sequencing with fluorescently labeled primers
For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
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.
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)
Cycle sequencing buffer (green cap)
Stop solution (purple cap)
95 % formamide containing EDTA, bromophenol blue, and xylene cyanol FF
PCR-grade water (white cap)
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.
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.
First prepare the following premix in a microcentrifuge tube:
|4 μl||10x Sequencing Buffer||green cap|
|1-2 pmol||fluorescent-labeled Primer||-|
|500-250 fmol or 30-150 ng/kb||DNA||-|
|1 μl||Sequencing Pol||red cap|
|fill up to 20 μl||PCR-grade water||white cap|
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:
|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|
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
Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463.