RT-PCR EasyTM II(Two Step)

Description

RT-PCR EasyTM II(Two Step) contains all the reagents for two-step RT-PCR, combining RT reaction and PCR reaction in the same kit, providing optimized RT Mix and PCR Mix. The unique RT Mix can synthesize high-quality first-strand cDNA easily and efficiently. 2× PCR EasyTM Mix Plus has high-efficiency and specific amplification. The organic combination of the two makes the detection of the target gene more sensitive.

Kit Contents

Features

• Real-time quantitative analysis of RNA can be carried out quickly and accurately.

• The kit uses a unique Foregene reverse transcription reagent and Foregene HotStar Taq DNA Polymerase combined with a unique reaction system to effectively improve the amplification efficiency and specificity of the reaction.

• The optimized reaction system makes the reaction have higher detection sensitivity, stronger thermal stability, and better tolerance.

Kit component information

2× RT EasyTM Mix:Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, reaction buffer, optimizer and stabilizer, etc.

2× PCR EasyTM Mix Plus :Foregene Taq DNA Polymerase, dNTPs, Mg2+,reaction buffer, optimizer and stabilizer.

Precautions: (Please read the precautions carefully before using the kit)

Reagents should avoid repeated freezing and thawing, otherwise the performance of the reagents will decrease or become invalid.

For templates, it is recommended to use RNA extracted from fresh samples or stored at -80℃ (RNA should avoid repeated freezing and thawing).

In order to avoid RNase contamination, the experiment operation should be carried out in the RNase-Free space; the pipette tips and PCR centrifuge tubes used must be RNase-Free; and disposable gloves and masks should be worn.

This kit must be used with specific primers for experiments. Please select the specific primers for the gene to be amplified according to the needs of the experiment.

Before use, put 2×RT EasyTM Mix and 2×PCR EasyTM Mix Plus on ice to completely melt, flick and mix well before use; the preparation of the system should be operated on an ice bath to improve the performance of the kit and improve Specificity of PCR amplification.

Precautions: (Please read the precautions carefully before using the kit)

• Reagents should avoid repeated freezing and thawing, otherwise the performance of the reagents will decrease or become invalid.

• For templates, it is recommended to use RNA extracted from fresh samples or stored at -80℃ (RNA should avoid repeated freezing and thawing).

• In order to avoid RNase contamination, the experiment operation should be carried out in the RNase-Free space; the pipette tips and PCR centrifuge tubes used must be RNase-Free; and disposable gloves and masks should be worn.

• This kit must be used with specific primers for experiments. Please select the specific primers for the gene to be amplified according to the needs of the experiment.

• Before use, put 2×RT EasyTM Mix and 2×PCR EasyTM Mix Plus on ice to completely melt, flick and mix well before use; the preparation of the system should be operated on an ice bath to improve the performance of the kit and improve Specificity of PCR amplification.

Transport and storage conditions

• Transportation conditions
  The whole process of low-temperature ice box transportation, to ensure that the kit is in a state of <4 °C.

• Storage conditions
  Stored at -20°C. Store the product in a constant temperature refrigerator at -20°C immediately after receipt. If the storage conditions are appropriate, the product will not degrade any performance during the 1-year validity period.

Template RNA concentration

RT-PCR EasyTM II(Two Step):(0.1pg-5μg total RNA)/20μl system

Operation guide

A-1:Preparation of materials and reagents

1. Prepare the prepared RNA template (it is recommended to use Foregene Total RNA Isolation Kit series kits to extract and purify RNA), specific primers (10μM) and related consumables and instruments.

Note: Please ensure the integrity of RNA and try to use RNA extracted from fresh samples.

2. Put 2×RT EasyTM Mix and RNase-Free ddH2O on an ice bath to let it melt naturally, and flick the tube wall to mix well for later use.

A-2:RT system preparation

2× RT EasyTM Mix is convenient and quick to use, avoiding pollution during operation and experimental errors caused by multiple preparations of the reaction system to the greatest extent. When using, just take half the volume of the reaction system (for example, if the reaction system is 20μl, take 10μl 2×RT EasyTM Mix solution), add RNA template and reverse transcription primers, and add RNase-Free ddH2O to make up the volume to 20μl. The specific RT reaction system preparation can refer to Table 1 below.

Form 1:RT system preparation

A-3:RT reaction program setting

1. After preparing the RT system with reference to the above table, mix gently (you can use a pipette tip to blow gently; you can also mix it on the vortexer and centrifuge it to collect the liquid scattered on the tube wall or tube cap, and place it in Ready to use on ice box).

2. Refer to the RT reaction program settings (Table 2) to set the reaction temperature, time, etc.

Note: In order to ensure the activity of Mix and improve its amplification efficiency, it is best to prepare the RT reaction system after the metal bath reaches the set reaction temperature (reverse transcription temperature 42℃ or 50℃), so that the system can be prepared immediately after the completion of the system preparation. Enter the reaction program. The RT reaction can also be performed on a PCR machine.

Form 2:RT reaction program setting

Note: When using Random primer, the reaction should be preheated at 25℃ for 10min before the first step of the reaction. The above procedure is for reference only. The actual reaction conditions vary depending on the structural conditions of the template, primers, etc. For RNA templates with complex secondary results, the first reaction temperature is recommended to be 50℃.

1. The reaction product can be directly used in subsequent tests, or stored at -20°C for up to a week. For long-term storage, it is recommended to avoid repeated freezing and thawing at -80°C.

B: PCR identification reaction

B-1:PCR reaction system preparation

Use the cDNA synthesized in step A as a template, and perform the PCR reaction with the 2×PCR EasyTM Mix Plus provided in the kit. Place 2×PCR EasyTM Mix Plus, cDNA, and RNase-Free ddH2O on an ice bath to let it melt naturally, and flick the tube wall to mix until use. For the specific PCR system preparation, refer to Table 3 below.

Form 3:PCR reaction system preparation

1*: Usually the final concentration of primer is 0.2-0.25μM to get better results. When the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1-0.5μM.

B-2:PCR reaction condition setting

1. After preparing the PCR system with reference to the above table, mix gently (you can use a pipette tip to blow gently; you can also mix it on the vortexer and centrifuge it to collect the liquid scattered on the tube wall or tube cap, and place it in Ready to use on ice box).

2. Refer to the PCR reaction program settings (Table 4) to set the temperature and time of the reaction.

Note: In order to ensure the activity of the PCR Mix and improve its amplification efficiency, it is best to set the PCR conditions on the PCR machine before preparing the PCR system.

Form 4:PCR reaction condition setting

Work Flow