One Step RT-qPCR SYBR Green Premix
Cat No: HCB5140A
One Step RT-qPCR Syber Green Premix is for fluorescence quantification based on SYBR Green I dye. Using gene-specific primers, reverse transcription and qPCR reactions are completed in one tube, eliminating the need for repeated cap-opening and pipetting operations, greatly improving assay efficiency and reducing the risk of contamination. For RNA samples, the kit employs heat-resistant Reverse Transcriptase for efficient cDNA synthesis and HotStart Taq DNA Polymerase for quantitative amplification. Under the optimized buffer system, the sensitivity of the kit can be as high as 0.1 pg for highly expressed targets and as high as 1 pg for moderately expressed targets. The kit is suitable for the amplification and quantification of DNA samples. It enables the sensitive detection and quantification of nucleic acids from different plant and animal samples, cells and microorganisms.
Components
|
No |
Name |
Volume |
Volume |
|
1 |
Advanced Buffer |
250 μL |
2×1.25 mL |
|
2 |
Advanced Enzyme Mix |
20 μL |
200 μL |
|
3 |
RNase Free H2O |
250 μL |
2×1.25 mL |
Storage Conditions
This product should be stored at -25~-15℃ away from light for 1 year.
Instructions
1.Reaction system configuration d
|
Components |
Volume (μL) |
Volume (μL) |
Final concentration |
|
Advanced Buffer |
12.5 |
25 |
1× |
|
Advanced Enzyme Mix |
1 |
2 |
- |
|
Forward Primer (10 μmol/L) a |
0.5 |
1 |
0.2 μmol/L |
|
Reverse Primer (10 μmol/L) a |
0.5 |
1 |
0.2 μmol/L |
|
RNA Tamplate b |
X |
X |
- |
|
RNase Free H2O c |
to 25 |
to 50 |
- |
Notes:
1) a. The final primer concentration was 0.2 μmol/L, which could also be adjusted between 0.1 and 1μmol/L as appropriate.
2) b. The reagent is extremely sensitive, with Total RNA in the range of 1pg-1μg, and testing of human samples showed an optimal input of 1 pg-100 ng, controlling for an overall Ct value in the range of 15-30 as appropriate.
3) c. It is recommended to use 20μL or 50μL to ensure the validity and reproducibility of target gene amplification.
4) d. Please prepare in the ultra-clean bench and use nuclease residue-free tips and reaction tubes; tips with filter cartridges are recommended. Avoid cross contamination and aerosol contamination.
2.Reaction program
|
Cycle step |
Temp. |
Time |
Cycles |
|
Reverse transcription |
50℃ a |
6mins |
1 |
|
Initial denaturation |
95℃ |
5mins |
1 |
|
Amplification reaction |
95℃ |
15 sec |
40 |
|
60℃ b |
30 sec |
||
|
Melting curve stage |
Instrument Defaults |
1 |
|
Notes:
1) a. The reverse transcription temperature can be selected between 50-55°C according to the experimental needs. For DNA samples, the reverse transcription process can be omitted.
2) b. In special cases the annealing/extension temperature can be adjusted according to the primer Tm value, 60°C is recommended.
Notes
1. This product is for research use only.
2. Please operate with lab coats and disposable gloves, for your safety.
