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(Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "The FastStart Essential DNA Green Master provides reagents, including SYBR Green I dye, for amplification and detection of DNA using the LightCycler^® 96 Instrument and the LightCycler^® Nano Instrument. The kit is ideally suited for hot start PCR assays for gene detection and quantification.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "The FastStart Essential DNA Green Master is function tested using the LightCycler^® System.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "Value": "Generation of PCR products can be detected by measurement of the SYBR Green I fluorescence signal. SYBR Green I dye intercalates into the DNA helix (Zipper, H., et al., 2004). In solution, the unbound dye exhibits very little fluorescence; however, fluorescence (wavelength, 530 nm) is greatly enhanced upon DNA binding. Therefore, during PCR, the increase in SYBR Green I fluorescence is directly proportional to the amount of double-stranded DNA generated. Since SYBR Green I dye is very stable (only 6% of the activity is lost during 30 amplification cycles) and the LightCycler^® 96 Instrument’s optics match the wavelengths of excitation and emission, it is the reagent of choice when measuring total DNA.
The basic steps of DNA detection by SYBR Green I during real-time PCR on the LightCycler^® 96 Systems are:

1. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal, which is subtracted during computer analysis.
2. After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules' light emission upon excitation.
3. During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls.
4. Fluorescence measurement at the end of the elongation step of every PCR cycle is performed to monitor the increasing amount of amplified DNA.

To prove that only your desired PCR product has been amplified, you may perform a melting curve analysis after PCR. In melting curve analysis, the reaction mixture is slowly heated to 95°C, which causes melting of double-stranded DNA and a corresponding decrease of SYBR Green I fluorescence. The instrument continuously monitors this fluorescence decrease and displays it as melting peaks. Each melting peak represents the characteristic melting temperature (Tm) of a particular DNA product (where the DNA is 50% double-stranded and 50% single-stranded). The most important factors that determine the Tm of dsDNA are the length and the GC-content of that fragment. If PCR generated only one amplicon, melting curve analysis will show only one melting peak. If primer-dimers or other nonspecific products are present, they will be shown as additional melting peaks. Checking the Tm of a PCR product can thus be compared with analyzing a PCR product by length in gel electrophoresis.

How this product works

FastStart Essential DNA Green Master is a ready-to-use reaction mix designed specifically for applying the SYBR Green I detection format in the LightCycler^® 480 Multiwell Plates 96, or LightCycler^® 8-Tube Strips on the LightCycler^® 96 Instrument. It is used to perform hot start PCR. Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR (Chou, Q., et al., 1992, Kellogg, D.E., et al., 1994, Birch, D.E., 1996) by minimizing the formation of nonspecific amplification products at the beginning of the reaction. FastStart Taq DNA Polymerase is a chemically modified form of thermostable recombinant Taq DNA polymerase that shows no activity up to 75°C. The enzyme is active only at high temperatures, where primers no longer bind nonspecifically. The enzyme is completely activated (by removal of blocking groups) in a single pre-incubation step (95°C, 5 to 10 minutes) before cycling begins. Activation does not require the extra handling steps typical of other hot start techniques.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "

Vial / Bottle	Cap	Label	Function / Description	Catalog Number	Content
1	green	FastStart Essential DNA Green Master, 2x conc.	Ready-to-use hot start PCR mix. Contains FastStart Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), SYBR Green I dye, and MgCl2.	06 402 712 001	5 vials, 1 ml each
06 924 204 001	10 vials, 5 ml each
2	colorless	FastStart Essential DNA Green Master, Water, PCR Grade	To adjust the final reaction volume.	06 402 712 001	5 vials, 1 ml each
06 924 204 001	2 vials, 25 ml each

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "Ready-to-use hot start reaction mix for real-time PCR with the LightCycler^® 96 System.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "Value": "Always run a negative control with the samples. To prepare negative controls:

• Replace template DNA with Water, PCR Grade (Vial 2; this will reveal whether a contamination problem exists).
• In a 2-step RT-PCR setup, omit addition of reverse transcriptase to the cDNA synthesis reaction (this will indicate whether DNA in RNA samples causes false-positive results).

", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "FastStart Essential DNA Green Master is a ready-to-use reaction mix using the SYBR Green I detection format on the LightCycler^® Nano and the LightCycler^® 96 Instrument. It is used to for hot-start PCR in 8-Tube Strips or single tubes. Hot-start PCR significantly improves the specificity and sensitivity of PCR by minimizing the formation of nonspecific amplification products at the beginning of the reaction.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "

Instruments and consumables

• LightCycler^® 96 Instrument*
  – Use with LightCycler^® 8-Tube Strips (white)* or LightCycler^® 480 Multiwell Plates 96 (white)* and LightCycler^® 480 Sealing Foils*
• Standard swinging-bucket centrifuge containing a rotor for multiwell plates with suitable adaptor
• Nuclease-free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement tips
• Sterile 1.5 ml reaction tubes for preparing master mixes and dilutions

Reagents for the LightCycler^® 96 Instrument*

• LightCycler^® Uracil-DNA Glycosylase* (optional)

For details about prevention of carryover contamination, see section Prevention of Carryover Contamination.

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "Variable, depending on the number of cycles and the annealing time. For example, if the cycling program specifies 45 cycles and an annealing time of 10 seconds and elongation time of 15 seconds, a LightCycler^® Nano PCR run will last about 53 minutes, including 10 minutes pre-incubation time, without melting curve. If the cycling program specifies 45 cycles with a 3-step protocol (10 second denaturation, 10 second annealing, 10 second elongation), a LightCycler^® 96 PCR run will last about 1:09 hours, including 10 minutes pre-incubation time, without melting.", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" } ] } } ] }