|
DNA Polymerases
Can another DNA Polymerases, other than the Minerva Biolabs' polymerases be used?
DNA polymerases that can be used include Taq, Tfl, and Tth. For a list of DNA polymerases that have displayed good results, please refer to our Download "Recommended Taqs".
Buffer
Can a separate buffer, other than the universal buffer supplied with the kit, be used?
It is possible to substitute the Venor® or the Onar®PCR 10x reaction buffer with the specific buffer supplied with the Taq DNA polymerase. However, the magnesium concentration of the buffer must then be adjusted
(e. g. to 3.0 mM in case of Venor®GeM and 4.0 mM Onar®Lp).
Primer/Nucleotide Mix
Can a separate Primer/Nucleotide mix other than the one supplied with the kit, be used?
The primers are essentially the core of our detection system. A modification would change the entire system. Furthermore, a self-made solution is time-consuming and can be unbalanced.
Internal and Positive Control DNA
The internal control is a cloned fragment containing the systems primer sequence. The positive control contains PCR amplicon DNA. The amplicon DNA in the positive control is provided in a lyophylized form. Rehydration of amplicon DNA is neither reproducable nor quantitative. Out of these reasons it is not possible to give a exact copy number for the positive control after rehydration and it can NOT be used for quantification.
For quantification purposes we offer quantification standards containing genomic DNA in solution (Cat.-No. 52-0XXX).
Thermalcycler
Which platforms have been used to develop the detection kits?
The QP kits where developed on the Roche LightCycler.
The DI kits where developed on the Stratagene SmartCycler.
Which platforms are compatible?
|
ABI 7000 SDS
ABI 7300 SDS
ABI 7500SDS
ABI 7700 SDS
ABI 7900 HT SDS
Bio-Rad iCycler
|
Cepheid SmartCycler
Corbett Rotor-Gene
MJ Opticon I & II
Roche Light-Cycler
Stratagene Mx3000p
Stratagene Mx4000
|
Which platforms are particular compatible with the 'DI' Detection Kits?
Smartcycler®; ABI Prism® 7000; Mx3000pT, Mx4000® & & related devices.
Which platforms are particular compatible with the 'QP' Detection Kits?
LightCycler®; OpticonT; ABI Prism® 7000, 7300, 7500, 7700, 7900;
Rotor-Gene & related devices.
Which are the specific modifications to the chemistry for a certain platform?
The chemical modifications refer exclusively to the primers, the "Scorpions".
Why do you deploy Scorpions primers in your quantitative diagnostic kits?
The Scorpions, labeled fluorogenic probes, are optimal for use in diagnostic assays and are compatible with most real-time quantitative PCR instruments.
We deploy two different types of Scorpions, hence two discriminative detection systems, the DI- and the QP-detection kit. By this means the customer can use various platforms to specifically perform his real-time quantitative Scorpions are used in real-time amplicon-specific detection of PCR products.
What is the generic composition/chemistry of Scorpions probes?
Scorpions probes contain an amplification primer linked, through an intermediary portion containing an amplification stopper, to a target specific probe. The sequences flanking the probe sequence are complementary to each other, and can form a hairpin structure. This hairpin structure brings a fluorophore and a quencher into close proximity.
During amplification, extension of the target sequence proceeds from the primer portion of the Scorpions probe. During denaturation, the hairpin loop opens. As the reaction cools following denaturation, the target-specific Scorpions probe sequence bind to the amplified, complementary target sequence, so the original hairpin structure does not re-form. Thus the fluorophore and quencher are separated, and fluorescence emission can be detected. The amplification stopper prevents read-through during amplification, which could lead to opening of the hairpin, and thus fluorescence detection, in the absence of target.
Because the Scorpions probe is integrated into the product, there is a direct relationship between the number of targets generated and the amount of fluorescence.
|
