TRzol (Total RNA Extraction Reagent) (Red)

The company's products are for scientific research purposes only and cannot be used for clinical diagnosis!

Product Introduction: TRzol reagent is a reagent that directly extracts total RNA from cells or tissues. It can maintain the integrity of RNA when breaking and dissolving cells. After adding chloroform and centrifuging, the sample was divided into a water sample layer and an organic layer. RNA exists in the aqueous layer. After collecting the water sample layer above, RNA can be recovered by isopropanol precipitation.

This method has good separation effects on small and large amounts of tissues and cells, whether they are human, animal, plant, or bacterial tissues. The simplicity of TRzol reagent operation allows for the simultaneous processing of multiple samples. All operations can be completed within one hour. The total RNA extracted by TRzol can avoid contamination of DNA and proteins. Therefore, it can perform RNA blot analysis, spot hybridization, poly (A)+selection, in vitro transcription, RNAse protection analysis, and molecular cloning.

TRzol reagent can promote the precipitation of various RNAs of different species and molecular weights. For example, RNA extracted from rat liver was electrophoretic on agarose gel and stained with ethidium bromide, and many discontinuous high molecular weight bands (mRNA and hnRNA components) between 7 kb and 15 kb, two dominant ribosomes~5kb (28S) and~2kb (18S), and low molecular weight RNA between 0.1 and 0.3 kb (tRNA, 5S) were seen. When the extracted RNA is diluted with TE, its A 260/A 280 ratio is ≥ 1.8.

Product storage: TRzol can be stably stored for 12 months at room temperature. However, for optimal results, we recommend storing in an environment of 2-8 ℃.

What reagents and equipment are required for PCR reaction?

Reagent:
Template DNA: PCR reactions require template DNA, such as extracting DNA from cells, tissues, blood, etc; Primers: Two primers used in PCR reactions that pair with specific regions required for amplification at both ends of the template DNA. Primers can also be synthesized into primers used in processes such as TA cloning; DNTPs: Four types of dNTPs are required in PCR reactions, including deoxyadenosine monophosphate (dATP), deoxythymidine monophosphate (dCTP), deoxyguanosine monophosphate (dGTP), and deoxycytosine monophosphate (dTTP), which are used in biological processes such as cell division and DNA synthesis, as well as for the extension and amplification of template DNA chains in PCR amplification; Taq DNA polymerase: a polymerase required for PCR reactions, generally using Taq polymerase, as well as other types of polymerases such as PFU, Phusion, etc., for amplifying DNA strands; PCR buffer solution: It has a buffering effect on the PCR reaction, adjusts the reaction pH, and can enhance the specificity of the PCR reaction by using hydrogen thiosulfate SDS and small molecule organic compounds such as formaldehyde, thereby improving reaction efficiency; Enzyme digestion system: PCR amplification often involves experimental steps such as recombination, construction, and sequencing, and often requires enzyme digestion operations. MARK: A molecular weight standard used to determine the size of DNA fragments. DNA Purification Kit: Used for purifying PCR reaction products;
equipment
PCR instrument: used to control the reaction temperature and ensure strict control of different temperature stages during PCR reaction; Electrophoresis tank: used for separating PCR amplification products; Nucleic acid extractor: Fully automatic extraction of nucleic acids from tissue samples. These reagents and equipment are used in PCR molecular biology technology, and only when they are ordered and complete can PCR reactions be carried out and accurate results be obtained.

PCR related basic experiments:

The basic steps of PCR reaction are generally composed of 20 to 35 cycles, each cycle including the following 3 steps:

1denaturation:

Separate double stranded DNA using high temperature (93-98 ℃). The high temperature will break the hydrogen bond connecting the two DNA strands. Before the first cycle, it is usually heated for a longer period of time to ensure that the template and primer are separated and only exist in single stranded form. This step takes 1-2 minutes, and then the PCR machine controls the temperature to enter the cycling stage.

2、 Annealing or bonding, annealing:

After DNA double stranded separation, lowering the temperature allows the primer to bind to single stranded DNA. The temperature at this stage is usually 5 ℃ lower than the melting point of the primer. Incorrect annealing temperature may result in primers not binding to the template or binding incorrectly. This step takes 1-2 minutes.

3、 Extension:

DNA polymerase starts synthesizing complementary strands along the DNA chain by binding to primers during cooling. The temperature at this stage depends on DNA polymerase. The timing of this step depends on the polymerase and the length of the DNA fragment to be synthesized. The traditional Taq method estimates the synthesis of 1000bp/min, while the newer Tbr (from the thermophilic bacterium Thermus brockianus) takes about 40 seconds, and the fusion polymerase produced by commercial companies only takes about 10-15 seconds.

Optimization of PCR reaction conditions:

1. Denaturation temperature and time:

Ensuring template DNA melting is the key to ensuring successful PCR amplification. Heating at 90-95 ° C for 30-60 seconds can cause even the most complex DNA molecules to transform into single strands. Excessive temperature or prolonged duration of high temperature can cause damage to Taq enzyme activity and dNTP molecules.

2. Restoration temperature and time:

The specificity of PCR amplification depends on the binding of primers and templates during the refolding process. The higher the annealing temperature, the higher the product specificity. The lower the annealing temperature, the lower the product specificity. Specific settings need to be made based on the Tm value of the primer.

3. Extended temperature and time:

Generally located between 70-75 ° C, which is the optimal temperature for Taq enzyme. When the primer is less than 16 nucleotides, excessive extension temperature is not conducive to the binding between the primer and the template. It can be slowly heated to 70-75 ° C. The extension reaction time can be determined according to the length of the amplified fragment, which is less than 1kb and 1 minute is sufficient; Exceeding 1kb requires an extended extension time. Taq enzyme can increase the time by 1kb/min. It should be noted that prolonged exposure may result in non-specific amplification. Therefore, it is necessary to set an appropriate extension time.

4. Number of cycles:

After selecting other parameters, the number of PCR cycles mainly depends on the concentration of template DNA. In theory, the accumulation of PCR products can reach its maximum value after 20-25 cycles. However, in practical operation, since the yield of each reaction step cannot reach 100%, regardless of the template concentration, 20-30 cycles are a reasonable number of cycles. The more cycles there are, the more non-specific amplification increases.

The products currently being sold by the company:

TRzol(Overall) RNA Extraction reagents)( red )	Pandemic2009H1N1 influenza virus HA gene nucleic acid detection kit	Coenzyme I NAD (H) Content Test Kit (Colorimetric Method)
TRzol(Overall) RNA Extraction reagents)( Colorless )	Seasonal influenza virus H3N2 subtype HA gene nucleic acid detection kit	NADKinase（NADK）Test box (colorimetric method)
TRzol LS(Liquid sample) RNA Extraction reagents)	H7N9 Subtype Virus HA Gene Nucleic Acid Detection Kit	Lactate dehydrogenase (LDH) test kit (colorimetric method)
RNApure Ultra pure total RNA Quick Extraction Kit (DNase I)	H7N9 Subtype Virus NA Gene Nucleic Acid Detection Kit	NAD malate dehydrogenase (NADMDH) test kit (colorimetric method)
EASYspin whole blood RNA Quick Extraction Kit (DNase I )	Influenza B virus nucleic acid testing kit	NADP malate dehydrogenase (NADPMDH) test kit (colorimetric method)
EASYspin organization / cell RNA Quick Extraction Kit (DNase I)	Yamagata strain nucleic acid testing kit for influenza B virus	Mitochondrial respiratory chain complex I/NADH coenzyme Q reductase test kit (colorimetric method)
EASYspin plant RNA Quick Extraction Kit (DNase I)	Victoria series nucleic acid testing kit for influenza B virus	NADH oxidase (NOX) test kit (colorimetric method)
PLANTS plant RNA Boosting agent	H7N9 variant virus (HPAI-H7) nucleic acid detection kit	Citrate synthase (CS) test kit (colorimetric method)
viral genome DNA/RNA Quick Extraction Kit	Seasonal influenza virus H1N1 subtype HA gene nucleic acid detection kit	Ethanol content test kit (colorimetric method)
RNAclean RNA Clean and purify reagent kit	Influenza C virus nucleic acid testing kit	Ethanol dehydrogenase（ADH）Test box (colorimetric method)
RNAfixer No liquid nitrogen RNA Sample storage solution	Eurasian avian and swine influenza virus (EA-H1N1) nucleic acid detection kit	Formaldehyde dehydrogenase (FDH) test kit (colorimetric method)
That's safe. efficient RNase inactivating agent	Influenza A virus/influenza B virus nucleic acid detection kit	Aldehyde dehydrogenase (ALDH) test kit (colorimetric method)
RNAlong RNA Long term storage solution	H7N9 Subtype Virus Nucleic Acid Test Kit/Contains Internal Standard	Coenzyme IINADP(H)Content testing box (colorimetric method)
RNase Away Ready to use and powerful RNase inactivating agent	Seasonal influenza virus H3N2 subtype nucleic acid detection kit	NADP phosphatase (NADPase) test kit (colorimetric method)