Product Introduction

Product Name	Cat. No.	Spec.
Universal One-Step RT-qPCR Kit	G3345-100	100 rxns

Product Description/Introduction

The product is a kit for direct real-time quantitative analysis of target RNA sequence by chimeric fluorescence method. In the same tube, RNA is first reverse transcribed to cDNA, and then qPCR reaction is carried out to avoid the risk of cross contamination between samples due to repeated opening of the lid. The thermal stability of the modified reverse transcriptase is significantly improved, which can effectively reverse the more complex templates. The reverse transcriptase premix also contains RNA inhibitors, which can effectively prevent the degradation of RNA from various sources. The one-step 2×RT-qPCR premix not only contains hot-start Taq DNA polymerase, dNTP, Mg2+ and other buffer components, but also has a unique ROX Passive Reference Dye, which is compatible with a variety of qPCR instruments. The premixture also contains a visual tracer blue dye to observe whether the reaction system is established or not, the spectrum of the blue dye does not overlap with the qPCR fluorescent dye and does not affect the reaction results.

Storage and Shipping Conditions

Ship with wet ice; store at -20℃, valid for 12 months.

Product Contents

Component Number	Component	G3345-100
G3345-1	RT Enzyme Mix	100 μL
G3345-2	2×Universal Blue One-Step RT-qPCR Mix	1 mL
G3345-3	Nuclease-Free Water	1 mL
Manual		One copy

Assay Protocol / Procedures

1. Reaction system (20 μL)

Component	Volume	Final Concentration
2×Universal Blue One-Step RT-qPCR Mix	10 μL	1×
RT Enzyme Mix	1 μL
Forward primer (10 µM)a	0.8 μL	0.4 μM
Reverse primer (10 µM)a	0.8 μL	0.4 μM
Template RNAb	Variable	<1 μg (Total RNA)
Nuclease-Free Water	To 20 μL

a. The final concentration of primers ranges from 0.2 to 1.0 μM, and the recommended concentration of primers is 0.4 μM.

b. The amount of template addition varies with the number of copies of target genes in the template solution. Gradient dilute the template and investigate the appropriate amount of template addition. Recommended template dosage is 0.1 ng-100 ng.

2. PCR reaction procedure (can be adjusted according to the model):

A. Two-step method					B. Three-step method
Stage	Step	Cycle	Temp	Time	Stage	Step	Cycle	Temp	Time
Stage 1	Reverse transcription	1	55°C	10 min	Stage 1	Reverse transcription	1	55°C	10 min
Stage 2	Pre- denaturation	1	95°C	1 min	Stage 2	Pre- denaturation	1	95°C	1 min
Stage 3	Denaturation	40	95°C	15 sec	Stage 3	Denaturation	40	95°C	15 sec
	Annealing /Extension		60°C	1 mina		Annealing		55-65°C	20 sec
						Extension		72°C	30 seca
Stage 4	Melting curve	1	Instrument default setting		Stage 4	Melting curve	1	Instrument default setting

a. To improve the specificity of amplification, the two-step procedure can be used or the annealing temperature can be increased. To improve the amplification efficiency, the three-step procedure can be used or the extension time can be extended.

Note

1. Before using 2×Universal Blue One-Step RT-qPCR Mix, gently turn it upside down and mix well, do not vortex to avoid bubbles.

2. When preparing the reaction solution, please place the reagent on the ice.

3. The product contains fluorescent dyes, so strong light should be avoided when preparing qPCR reaction solution.

4. New disposable tips should be used for preparation of reaction mixes to avoid cross contamination.

5. To ensure the success of the reaction, please use a high quality RNA template. Repeated freeze-thaw RNA templates will affect amplification. If many experiments are needed, it can be frozen after sub-packing to reduce the number of freezing and thawing.