4X One Step Fast RT-qPCR Premix-UNG (one-tube) HYA435
One Step Fast RT-qPCR Premix-UNG is a highly stable one-tube probe-based mix suitable for one- step reverse transcription and quantitative PCR (qRT-PCR). It supports pre-mixing of primers and probes and stay stable after long time storage at low temperature. The sample to be tested can be added directly when using, without additional tube opening/pipetting operation. This product provides components, e.g. hot-start DNA polymerase, M-MuLV, heat-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl2, dNTPs (with dUTP instead of dTTP), and stabilizers. With the genetically modified rapid amplification reverse transcriptase and DNA polymerase, it is possible to complete the PCR amplification within 20~40 minutes. This reagent uses special buffer for qPCR
with mixed enzymes of anti-inhibitory amplification enzyme and UNG enzyme. Therefore, it can obtain good amplification of target genes and prevent false amplification caused by PCR residual and aerosol contamination. This reagent is compatible with most fluorescence quantitative PCR instruments from manufacturers such as Applied Biosystems, Eppendorf, Bio-Rad and Roche. This product has been treated by special process without DNA residue, which can be used for the development of biopharmaceutical related detection reagents.
Components
4X One Step Fast RT-qPCR Premix-UNG (one tube)
Storage
All components should be kept at -20℃ for long-term storage and 4℃ for up to 3 months. Please mix thoroughly after thawing and centrifuge before using. Avoid frequent freeze-thaw.
Instructions
1. Reaction system
|
Components |
25 uL Volume |
50 uL Volume |
Final Concentration |
|
4X One Step Fast RT-qPCR Premix-UNG (one tube) |
6.25 µL |
12.5 µL |
1× |
|
25×Primer-Probe Mix* |
1 uL |
2 uL |
1× |
|
Template RNA** |
- |
- |
- |
|
DEPC H2O |
Up to 25 µL |
Up to 50 µL |
- |
*The final concentration of primer is usually 0.2 μ M. For better results, the primer concentration can be optimized within the range of 0.2-1 μM. Generally, the probe concentration can be optimized within the range of 0.1-0.3 μM. When using a fast PCR Procedure, increasing the concentration of primers and probes may result in better amplification results, and their ratio should be optimized accordingly.
**Different types of samples contain different types and content of inhibitor and copy number of target gene. The sample volume should be considered by actual condition. Make a dilution of the sample with nuclease-free water or TE Buffer, if necessary.
2. Reaction program
|
Regular PCR program |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Reverse transcription |
50℃ |
10-20 min |
1 |
|
Polymerase activation |
95℃ |
1-5 min |
1 |
|
Denaturation |
95 ℃ |
10-20 s |
40-50 |
|
Annealing and Extension |
56~64℃ |
20-60 s |
|
|
Rapid PCR program |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Reverse transcription |
50℃ |
5 min |
1 |
|
Polymerase activation |
95℃ |
30 s |
1 |
|
Denaturation |
95 ℃ |
1-3 s |
40-50 |
|
Annealing and Extension |
56~64℃ |
3-20 s |
|