Reverse Transcription PCR Two-Step (RT-PCR) Kit Product Manual

Reverse Transcription PCR Two-Step Method ( RT-PCR ) Kit Product Manual (Chinese version)

The main purpose

The reverse transcription PCR two-step (RT-PCR) reagent is a method for analyzing RNA by reverse-transcription of RNA into cDNA by reverse transcriptase and then in vitro amplification of DNA (PCR method). Constructing an authoritative and classic technical approach. The technology has been carefully developed and successfully tested. It is suitable for RNA analysis, cDNA cloning, RNA expression and the like. Especially useful for low copy RNA samples. The product is ready to use, stable in performance, strictly free of nuclease contamination, easy to operate, high in reverse transcription efficiency and amplification yield, and good in repeatability.

technical background

Single-stranded DNA Binding Protein (SSB) is used to improve the reaction product and efficiency of reverse transcriptase. Super reverse transcriptase is a DNA polymerase used to synthesize paired DNA with single-stranded RNA as a template and maximize the yield of cDNA in the first strand. RNA nuclease inhibitors (RNAsin) have a broad spectrum of stable RNase inhibition functions for high quality clearance of RNase contamination.

product content

Guide Liquid (Reagent A) 90 μl

Reverse Solution (Reagent B) 162 μl

Reaction solution (Reagent C) 600 μl

Replenishing solution (Reagent D) 2 ml

Product manual 1 copy

storage method

Store in a -20 ° C refrigerator to avoid repeated freezing and thawing; effective to ensure June

User-supplied

RNA sample: template for reverse transcription amplification

PCR primers: promoter DNA fragments for reverse transcription

Dry thermostat: incubation for reverse transcription reactions

0.5 ml PCR tube: container for reverse transcription and PCR reactions

Vortex Oscillator: For reactant mixing

Micro Tabletop Centrifuge: for sample handling

PCR instrument: for nucleic acid product amplification

Experimental procedure

Before the start of the experiment, the reagent solution (Reagent A), reverse reagent (Reagent B), reaction solution (Reagent C) and replenisher (Reagent D) in the kit at -20 °C in the refrigerator, and user-supplied RNA samples The primers were placed in an ice bath and melted; the dry thermostats were set to 70 ° C and 37 ° C, respectively. Then do the following.

The first step: cDNA synthesis

• Vortex the reagent solution in the melted kit for 2 seconds
• Place in a micro tabletop centrifuge for 5 seconds of centrifugation
• Remove 4.5 μl of Guide Liquid (Reagent A) into a new 0.5 mL PCR tube
• Add 8 μl of user-supplied RNA sample (5 μg total RNA or 0.25 μg POLY A+ RNA)
• Mix with the gun
• Incubate for 3 minutes at 70 ° C dry thermostat
• Immediately put into the ice trough
• Add 8 μl of reverse solution (Reagent B)
• Mix with the gun
• Place in a micro tabletop centrifuge for 5 seconds of centrifugation
• Incubate for 60 minutes at 37 ° C dry thermostat
• Immediately put into the ice trough
• Add 80 μl of replenisher (Reagent D) to dilute and mix.
• Put it in the ice trough or put it in the refrigerator at -20 °C.

Step 2: cDNA amplification

• Remove 30 μl of reaction solution (Reagent C) into a new 0.5 mL PCR tube
• Add user-prepared primers F and R (total of 350 ng, respectively)
• Add 2 μl of the above diluted cDNA composition
• Finally add the rehydration solution (Reagent D) to a total of 50 μl, mix well
• Centrifuge for 5 seconds in a 4°C micro tabletop centrifuge
• Add 2 drops of mineral oil using a 1000 μl pipette tip (note: see note 6 )
• Put into the PCR instrument, the PCR reaction procedure is:

• The PCR product was stored in a -20 ° C freezer until any subsequent experiments were performed.
• Agarose gel electrophoresis analysis

Precautions

• This product is 20 operations
• The entire operation must be carried out at 4 ° C
• Avoid repeated freezing and thawing
• Gloves are required for the entire operation. It is recommended to use the filter tip and take extra care to prevent degradation of RNA.
• It is recommended that all operating items be RNA-specific
• It is recommended to use 5 micrograms of total RNA sample volume; to ensure satisfactory results, do not lower than 500 nanograms
• According to the performance of the user's PCR instrument, decide whether to add mineral oil (Mineral Oil)
• Determine the Tm temperature according to the formula of Tm=4(G+C)+2(A+T); it is recommended to use analysis software.
• The company provides a series of RT-PCR products

Quality Standard

• This product has been certified to be stable.
• This product has been certified to be strictly free of nuclease contamination
• This product has been identified and has a high yield.

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