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.
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
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-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.
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|4 µl||10x Sequencing Buffer||green cap|
|1-2 pmol||fluorescent labelled Primer||-|
or 30-150 ng/kb
|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|
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
Overview PCR Master Mixe:
BioStar 2 PCR mastermix with SYBRGreen blue dye, no rox
BioStar 6 PCR mastermix
for probes 60 mM LOW-ROX
BioStar 7 PCR mastermix
for probes 900 mM HIGH-ROX
DNA Cycle Sequening Kit