Virus Genomic DNA/RNA Rapid Extraction Kit

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PCRBasic steps and precautions?

1、 Experimental principle

PCRIt is an in vitro artificial selective amplificationDNAA method similar to in vivoDNACopy it. in vivoDNACopying requires templates, primersDNAPolymeraseDNAHelicasedNTPsAnd outside the bodyPCRThe reaction also requires similar components, including templates, primersPCR BufferTheTaqEnzymesdNTPsPrimers are artificially designed specific sequences that achieve amplification at specific positions;PCR BufferProvide a buffering environment for reactions; The reaction process is the same as in living organisms,DNADouble strand opening, primer binding to template, extending to form a new strand. And these processes rely onDNAEnzymatic hydrolysis of chains is achieved in vitro by controlling the reaction temperature. As commonly used94℃Transgender TemplateDNAOpen the double strand, bind the primer to the template at annealing temperature, and finally72℃Complete the extension and repeat this process repeatedly to achieve specific fragmentsDNAA large amount of amplification. It wasn't until the third cycle that the target was generatedDNASame sectionDNAMolecules further cyclically generate targetsDNADouble the index of the segment.

In the later stage of amplification,Due to product accumulation,Transform the originally exponentially amplified reaction into a flat curve,The product no longer increases significantly with the number of cycles,This is called the platform effect. plateau period(Plateau)It will continue to amplify the low concentration non-specific products that were originally generated due to mismatches, reaching a higher level. Therefore, the number of cycles should be appropriately adjusted to end the reaction before the plateau period and reduce non-specific products. Arriving at the platform period(Plateau)The required number of cycles depends on the copy of the template in the sample.

2、 Main components

1.Templates can take various forms, mainly including genomesDNAPlasmidsDNAGenome carrying virusesDNAThePCRProduct,cDNAWait, but not forRNAFor different types of templates, the main differences lie in the time of pre denaturation and the amount of templates. Generally for large genomesDNAPre denaturation time10minEnough, plasmidDNA2minGenome carrying virusesDNAPre denaturation2minThePCRProduct pre denaturation2minJust enough.

AttentioncDNAFor single chainDNABut it can still be donePCRThe template only binds to one primer in the first cycle to synthesize another chain. Starting from the second round, both primers bind to specific sites, thus achieving compatibility with conventional primersPCRThe integration. And as a single chainRNABut it cannot be carried outPCRAmplification, the reason lies in the implementationPCRThe reaction isDNAPolymerase, can only be specifically recognizedDNAChain.

2.For the quantity of templates, generally25ulsystemDNAThe quality is50100ngRegarding the genomeDNADue to its complex structure, the extracted concentration is often high. To prevent excessive concentration from affectingPCRCausing impact, therefore it is necessary to extractDNAPerform gradient dilution. Otherwise, excessive concentration may cause non-specific amplification. For plasmids andPCRDue to its simple structure and generally low extraction concentration, the product does not require dilution.

PCRWhat are the issues that should be noted in experiments?

nonectvalue

Did you encounter any test resultsCtValue situation,Check if there are any of the following issues:

1Insufficient number of cycles(Generally not exceeding45cycle,Not only does the background value increase,Quantification is also inaccurate);

2ThePCRProgram setting error,The steps for detecting fluorescence signals are incorrect. generalSGLaw adoption72℃Collection during extension,TaqManThe rule usually collects signals at the end of annealing or during extension,Also, is fluorescence collection selected;

3Primer or probe degradation. can pass throughPAGEElectrophoretic detection of whether primers and probes degrade;

4The template size may degrade or the sample size may be insufficient(not exceed500ng,Just follow the instructions of the reagent kit),For samples with unknown concentrations, the highest concentration of the series of diluted samples should be used as the starting point;If template degradation occurs,Consideration should be given to the introduction of impurities and repeated freeze-thaw cycles in sample preparation,Suggest to pack template samples in small quantities for storage,Avoid repeated freeze-thaw cycles;

5Is the primer probe suitable(Especially when primers cross introns,To ensure the amplification of the genomeDNA;Upstream and downstream primersTmValue exceeds4℃The above will also affect amplification).

ctValue too late

In relative quantification, CtThe value is generally controlled within1525It's better between them,If in absolute quantification,For low copy number samples,CtThe value will increase,But generally it should not exceed40cycle,Otherwise, the quantification will be inaccurate.

Therefore,judgmentCtIs it considered abnormal if the value appears too late,It needs to be based on specific experimental design and objectives.

1The amplification efficiency is low. Inappropriate ratio between primers or between primers and probes,Optimization is needed;Unreasonable primer or probe design,Need to redesign;

2ThePCRInappropriate program,Switching to a three-step reaction method,Or optimize annealing/Extended temperature,The annealing temperature can be appropriately reduced;annealing/Short extension time(Can be extended under recommended time conditions10s);

3TheMgCl2Inappropriate concentration,Increase the concentration of magnesium ions, etc.PCRDegradation of various reaction components or insufficient sample size;

4ThePCRThe product is too long.PCRProduct design exceeds500bp;

5There are inhibitors present in the template. Using high-purity templatesPCRDetect or dilute the template.

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