Description
HifairTM Lyo-Ready I Multiplex One Step RT-qPCR Kit（UDG plus) is a multiplex quantitative PCR kit based on RNA as template. In the process of the experiment, reverse transcription and quantitative PCR were carried out in the same tube, which simplified the experimental operation and reduced the risk of contamination.The unique Buffer and enzyme design ensures that it can be used in the preparation and product design of one-step RT-qPCR lyophilized reaction systems.
In this kit, the first strand cDNA was efficiently synthesized by heat-resistant HifairTM Reverse Transcriptase and quantitatively amplified by UniconTM HotStart Taq DNA Polymerase. The kit mainly contains optimized lyophilizable buffer, enzymes mix, etc. In addition, the factors that can effectively inhibit the non-specific PCR amplification and improve the amplification efficiency of multiple qPCR reactions are added, which can ensure the amplification efficiency and carry out up to multiple amplification reaction.
This kit is fitted with lyoprotectant that can be used for lyophilization.
Specifications
|
Components No. |
16645ES60 / 16645ES80 / 16645ES92 |
|
Size |
100 T / 1000 T / 10000 T |
Components
|
Name |
16645ES60 (100T) |
16645ES80 (1,000T) |
16645ES92 (10,000T) |
|
|
16645-A |
4×HifairTM Lyo-Buffer |
600 μL |
6 mL |
60 mL |
|
16645-B |
HifairTM Lyo-Enzyme Mix |
100 μL |
1 mL |
10 mL |
|
16645-C |
Lyoprotectant |
800 μL |
8 mL |
80 mL |
Storage
The product should be stored at -25℃ ~ -15℃ for 1 year. It should avoid repeated freezing and thawing.
Instructions
1. Reaction Composition:
|
Components |
Volume (μL) |
Final Concentration |
|
4×HifairTM Lyo-Buffer |
6 |
1× |
|
HifairTM Lyo-Enzyme Mix |
1 |
- |
|
Lyoprotectant |
8 |
- |
|
Primer Mix (10 μM) |
1 |
|
|
Probe Mix (10 μM) |
0.5 |
0.05-0.5 μM |
|
Template RNA |
5 |
- |
|
DEPC H2O |
to 25 |
- |
Note：Be sure to mix well before use, avoid excessive bubbles caused by violent vibration.
2. Optimized Cycling Protocol
(1) Standard amplification procedure
|
Reaction stage |
Temperature |
Time |
Cycle |
|
|
1 |
Reverse transcription |
50°Ca |
1 |
|
|
2 |
Initial denaturation |
95°C |
5 min |
1 |
|
3 |
Amplification reaction |
95°C |
|
45 cycles |
|
60°Cb |
30 secc |
(2) Rapid amplification program
|
|
Reaction stage |
Temperature |
Time |
Cycle |
|
1 |
Reverse transcription |
50°Ca |
2 min |
1 |
|
2 |
Initial denaturation |
95°C |
2 sec |
1 |
|
3 |
Amplification reaction |
95°C |
|
45 cycles |
|
60°Cb |
13 secc |
Note：
a) Reverse transcription: The temperature can select 42°C or 50°C for 10-15 minutes.
b) Amplification reaction: The temperature is adjusted according to the Tm value of the designed primers.
c) Fluorescence signal acquisition: Please set the experimental procedure according to the requirements of the instrument manual.
3. Application equipment
Equipment with Rox：ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™, StepOne Plus™
Equipment with Low Rox：ABI 7500, 7500 Fast, ViiA™7, QuantStudio™ 3 and 5, QuantStudio™ 6,7,12k Flex
Stratagene MX3000P™, MX3005P™, MX4000P™
Equipment without Rox：
Bio-Rad CFX96™, CFX384™, iCycler iQ™, iQ™5, MyiQ™, MiniOpticon™, Opticon®, Opticon® 2, Chromo4™
Eppendorf Mastercycler® ep realplex, realplex 2 s; Qiagen Corbett Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene® 6000
Roche Applied Science LightCycler® 480, LightCycler® 2.0, Lightcycler® 96
Thermo Scientific PikoReal Cycler; Cepheid SmartCycler®; Illumina Eco qPCR
Notes
Please wear the necessary PPE, such lab coat and gloves, to ensure your health and safety！
Please use RNasefree consumables for the experiment.
This product is for research use only！
Lyophilization procedure
This kit can be directly used for lyophilization, or you can further explore excipients for different primers on this basis. The standard lyophilization procedure of this reagent is as follows:
|
Process |
temperature |
set time |
duration |
Vacuum mbar |
|
prelyophilization |
-45℃ |
10 min |
60 min |
—— |
|
Initial freeze-drying stage |
-45 ℃ |
10min |
60min |
0.01mbar |
|
-40 ℃ |
10min |
900min |
0.01mbar |
|
|
-35 ℃ |
10min |
60min |
0.01mbar |
|
|
-30 ℃ |
20min |
60min |
0.01mbar |
|
|
-20 ℃ |
20min |
60min |
0.01mbar |
|
|
0 ℃ |
20min |
60min |
0.01mbar |
|
|
pressure built-up test |
||||
|
Analytical drying stage |
5 ℃ |
10min |
20min |
0.01mbar |
|
10 ℃ |
10min |
20min |
0.01mbar |
|
|
15 ℃ |
10min |
20min |
0.01mbar |
|
|
20 ℃ |
10min |
20min |
0.01mbar |
|
|
25 ℃ |
10min |
240min |
0.01mbar |
|
|
pressure built-up test |
||||