| DNA Polymerases
We highly recommend our reliable hot-start MB Taq DNA Polymerase (Cat.
No. 53-0200) for optimal performance and most sensitive results. We cannot
guarantee excellent results with other polymerases.
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 'qEP' Detection Kits?
| Instrument |
Type 1 |
Type 2 |
| LightCycler® 1.2 |
+v |
- |
| LightCycler® 1.5 |
+ |
- |
| LightCycler® 2.0 |
+ |
o |
| LightCycler® 480 |
+ |
o |
| Rotor-GeneTM 3000 |
+ |
o |
| Rotor-GeneTM 6000 |
+v |
o |
| ABI Prism® 7000 |
- |
+ |
| ABI Prism® 7300 |
- |
+ |
| ABI Prism® 7500 |
- |
+v |
| ABI Prism® 7700 |
- |
+ |
| ABI Prism® 7900 |
- |
+ |
| iCycler iQ® |
o |
o |
| iQ™5 |
o |
o |
| Opticon 2 |
- |
o |
| Chromo 4 |
o |
o |
| MX3000P® |
o |
o |
| MX4000® |
o |
o |
+ = recommended kit version
– = not compatible
o = untested but presumed to be compatible
V = validated
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.
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