RNAclean RNA Purification Kit

PCRWhat reagents and equipment are required for the reaction?

reagent:
templateDNA：PCRResponse requires a templateDNA, such as extracting from cells, tissues, and bloodDNAWait; Primers:PCRTwo primers for the reaction, one with the templateDNAThe specific region required for paired amplification at both ends can also be synthesized as primersTAPrimers used in cloning and other processes;dNTPs：PCRFour types are required in the reactiondNTPs, including deoxyadenosine monophosphate（dATP）Deoxythymidine acid（dCTP）Deoxyguanosine monophosphate（dGTP）Deoxycytosine acid（dTTP）, used for cell divisionDNABiological processes such as synthesis, used forPCRExpanded templateDNAExtension and amplification of chains;Taq DNAPolymerase:PCRThe polymerase required for the reaction is generally usedTaqPolymerase, as well as some other types of polymerases such asPFUThePhusionWait, used for amplificationDNAChain;PCR bufferSolution: YesPCRThe reaction has a buffering effect and regulates the reactionpHThiosulfate hydrogen saltSDSSmall molecule organic compounds such as formaldehyde can enhancePCRReaction specificity, thereby improving reaction efficiency; Enzyme digestion system:PCRAmplification often involves experimental steps such as recombination, construction, and sequencing, and often requires enzyme digestion operations.MARK:A molecular weight standard used to distinguishDNATool for fragment size.DNAPurification kit: used for purificationPCRReaction products;
equipment
PCRInstrument: used to control the reaction temperature and ensurePCRStrict control of different temperature stages during the reaction; Electrophoresis tank: used for separationPCRAmplification product; Nucleic acid extractor: Fully automatic extraction of nucleic acids from tissue samples. These reagents and equipment arePCRIn molecular biology techniques, only on the basis of order and completeness can it be carried outPCRReact and obtain accurate results.

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

PCRRelated basic experiments:

PCRThe basic steps of a polymerase chain reaction are generally as follows:20to35Composed of cycles, each cycle includes the following3Step by step:

1、 Transgender:

Utilize high temperature（93-98℃）Make double chainDNASeparate. High temperature will connect two linesDNAThe hydrogen bonds of the chain are broken. 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. The time for this step1-2Minutes, nextPCRThe instrument controls the temperature to enter the cycling stage.

2、 Annealing or bonding,Restoration:

inDNAAfter double strand separation, lowering the temperature allows the primer to bind to the single strandDNAUp there. The temperature at this stage is usually lower than the melting point of the primer5℃Incorrect annealing temperature may cause primers to not bind to the template or to bind incorrectly. The time for this step1-2minute

3、 Extension:

DNAThe polymerase starts from the primer bound during cooling and follows theDNASynthesize complementary chains. The temperature at this stage depends onDNAPolymerase. The timing of this step depends on the polymerase and the synthesis requiredDNAFragment length. traditionalTaqEstimated synthesis1000bp/minNewerTbr(From thermophilic bacteria)Thermus brockianus）About40The fusion polymerase produced by commercial companies only requires about10-15In seconds.

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PCROptimization of reaction conditions:

1Denaturation temperature and time:

Ensure templateDNAChain breaking is to ensure the entire processPCRThe key to successful amplification. heat90~95°C, 30~60sNo matter how complex it isDNAMolecules can also transform into single chains. If the temperature is too high or the duration of high temperature is too long, it can be harmfulTaqEnzyme activity anddNTPMolecules cause damage.

2Restoration temperature and time:

PCRThe amplification specificity 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. According to the primerTmSpecific value settings.

3Extension temperature and time:

Generally locatedTaqThe optimal temperature for enzyme action70~75°Cbetween. Primer less than16When it comes to nucleotides, excessively high extension temperature is not conducive to the binding of primers and templates, and can be slowly heated up to70~75°CThe extended reaction time can be determined based on the length of the fragment to be amplified, which is less than1kb, 1minSufficient; greater than1kbNeed to extend the extension time.TaqEnzymes can be used according to1kb/minIncrease time. It should be noted that prolonged exposure may result in non-specific amplification. Therefore, it is necessary to set an appropriate extension time.

4Number of cycles:

After selecting other parameters,PCRThe number of cycles mainly depends on the templateDNAThe concentration. theoretically speaking20~25After the second cycle,PCRThe accumulation of products can reach the maximum value, but in practical operation, it is impossible to achieve the yield of each reaction step100%Therefore, regardless of the template concentration,20~30This is a reasonable number of cycles. The more cycles there are, the more non-specific amplification increases.