If you wanted to prepare only one PCR reaction, how much of each reagent would you add to the PCR tube?]What is the final volume of the individual PCR reactions we are making? D. Retrigeration• If the thermal cycler has a refrigeration or "soak" cycle, the cycling reaction canbe programmed to end by holding the tubes at 4°C for several hours.• This cycle can minimize any polymerase activity that might occur at highertemperatures, although this is not usually a probiem.E. Cycle NumberGenerally, 25-30 cycles result in optimal amplification of desired products.Occasionally, up to 40 cycles may be performed, especially for detection oflow-copy targets.1. Standard ApplicationReagents to be Supplied by the Usertemplate DNAupstream primer1. Thaw the Go Taq Green Master Mix at room temperature. Vortex the Master Mix,then spin it briefly in a microcentrifuge to collect the material at the bottom of thedownstream primermineral oil (optional)tube.2. Prepare one of the following reaction mixes on ice:For a 25µl reaction volume:ComponentGoTaq® Green Master Mix, 2Xupstream primer, 10uMdownstream primer, 10LMII. General ConsiderationsVolumeFinal Conc.1X12.5pl0.25-2.5ul0 25-25ul1-5ul25ul0.1-1.0uM01-1.0uM

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If you wanted to prepare only one PCR reaction, how much of each reagent would you add to the PCR tube?]What is the final volume of the individual PCR reactions we are making? D. Retrigeration• If the thermal cycler has a refrigeration or &#34;soak&#34; cycle, the cycling reaction canbe programmed to end by holding the tubes at 4°C for several hours.• This cycle can minimize any polymerase activity that might occur at highertemperatures, although this is not usually a probiem.E. Cycle NumberGenerally, 25-30 cycles result in optimal amplification of desired products.Occasionally, up to 40 cycles may be performed, especially for detection oflow-copy targets.1. Standard ApplicationReagents to be Supplied by the Usertemplate DNAupstream primer1. Thaw the Go Taq Green Master Mix at room temperature. Vortex the Master Mix,then spin it briefly in a microcentrifuge to collect the material at the bottom of thedownstream primermineral oil (optional)tube.2. Prepare one of the following reaction mixes on ice:For a 25µl reaction volume:ComponentGoTaq® Green Master Mix, 2Xupstream primer, 10uMdownstream primer, 10LMII. General ConsiderationsVolumeFinal Conc.1X12.5pl0.25-2.5ul0 25-25ul1-5ul25ul0.1-1.0uM01-1.0uM<250ngNA.A. GoTaq® Green Master Mix CompatibilityGoTaqe Green Master Mix is compatible with common PCR additives such as DMSO andbetaine. These additives neither change the color of GoTaq® Green Master Mix nor affectdye migration.DNA templateNuclease-Free Water toIf bath agarose gel analysis and further downstream applications involving absorbance arfluorescence will be used, the two dyes can be removed from reactions using standardPCR clean-up systems such as the Wizarde Sv Gel and PCR Clean-Up System (CatAS281).For a 50pl reaction volume:ComponentGoTaq Green Master Mix, 2Xupstream primer, 10uMdownstream primer, 10uMDNA templateNuclease-Free Water toVolumeFinal Conc.25ul05-5.0ul0.5-5.Qul1-5ul50ul1X0.1-1.QuM0.1-1.0uM<250ngB. Primer DesignPCR primers generally range in length from 15-30 bases and are designed to flank theregion af interest. Primers should contain 40-60% (G + C), and care should be taken toavoid sequences that might produce internal secondary structure The 3-ends of theprimers should not be complementary to avoid the production of primer-dimers. Primer-dimers unnecessarily deplete primers from the reaction and result in an unwantedpolymerase reaction that competes with the desired reaction. Avoid three G or Cnuclectides in a row near the 3-end of the primer, as this may result in nonspecificprimer annealing, increasing the synthesis af undesirable reaction products. Ideally, bothprimers should have nearly identical melting temperatures (T in this manner, the bwoprimers should anneal roughly at the same temperature. The annealing temperature of thereaction is dependent upon the primer with the lowest Ta. For assistance with calculatingthe Tm of any primer, a m Calculator is provided on the BioMath page of the Promegaweb site at: www.promega.com/biomath/NAFor a 100pl reaction volume:ComponentGoTaq Green Master Mix, 2Xupstream primer, 10uMdownstream primer, 10uMDNA templateVolumeFinal Conc.50ul1X1.0-10.0gl0.1-1.0gM0.1-1.0uM<250ngNA1-5ul100ulNuclease-Free Water to3. If using a thermal cycler without a heated lid, overlay the reaction mix with 1-2 drops(approximately 50ul) of mineral oil to prevent evaporation during themal cycling.Centrifuge the reactions in a microcentrituge for 5 seconds.4. Place the reactions in a themal cycler that has been preheated to 95°C. Perform PCRusing your standard parameters.C. Amplification TroubleshootingTo overcome low yield or no yield in amplifications (eg, mouse tail genotyping2pplications), we recommend the following suggestions:• Adjust amealing temperature. The reaction buffer compositian affects the metingproperties of DNA. See BioMath Calculator to calculate the melting temperature forprimers in the GoTage reation (www.promega.com/biamalhy.II. General Guidelines for Amplification by PCRA. DenaturationGenerally, a 2-minute initial denaturation step at 95°C is sufficient.Subsequent denaturation steps will be between 30 seconds and 1 minute.Minimize the effect of amplifiction inhibitors. Some DNA isolation procedures,particuiarly genomic DNA isolation, can result in the copurification of amplificationinhibitors. Reduce the valume of template DNA in reaction or dilute template DNAprior to adding to reaction. Diluting samples even 1:10,000 has been shown to beeffective in improving results, depending on initial DNA cancentration.• Increase template DNA purity. Include an ethanoi precipitation and wash step prior toamplification to remove inhibitors that copurify with the DNA• Add PCA additives. Adding PCR-enhancing agents (e.g. DMSO or betaine) mayimprove yields. General stabilizing agents such as BSA (Sigma Cat A7030; finalconcentration 0.16mg/ml) also may help to overcome amplification tailure.B. Annealing• Optimize the annealing conditions by performing the reaction startingapproximately 5°C below the calculated melting temperature of the primers andincreasing the temperature in increments of 1°C to the annealing temperature.• The annealing step is typically 30 seconds to 1 minute.C. Extension• The extension reaction is typically performed at the optimal temperature forTaq DNA polymerase, which is 72-74°C.• Allow approximately 1 minute for every 1kb of DNA to be amplified.• Afinal extension af 5 minutes at 72-74°C is recommended.P. Mere Information on Amplification Certificate of AnalysisGoTaq® Green Master MixPart# 9PIM712Cat.#M7121Size10 reactionsRevised 4/18M7122100 reactionsM7123M712B-C1,000 reactions1x1,250ulCat.# M7121, M7122 and M7123 include GoTaq® Green Master Mix, 2X, and Nuclease-Free Water.Cat.# M712B-C does not include Nuclease-Free Water.Description: GoTaq® Green Master Mixla.b) is a premixed ready-to-use solution containing bacterialy derived Taq DNApolymerase, DNTPS, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA termplates by PCR.GoTaq® Green Master Mix contains two dyes (blue and yellow) that allow monitoring of progress during electrophoresis.Reactions assembled with GoTaq® Green Master Mix have sufficient density for direct loading onto agarose gels.AF9PIM712 0418M712GoTaq® Green Master Mix is recommended for any amplification reaction that will be visualized by agarose gel electrophoresisand ethidium bromide staining. The master mix is not recommended if any downstream applications use absorbance orfluorescence excitation, as the yellow and blue dyes in the reaction buffer may interfere with these applications. The dyes absorbbetween 225-300nm, making standard A2so readings to determine DNA concentration unreliable. The dyes have excitationpeaks at 488nm and between 600-700nm that correspond to the excitation wavelengths commonly used in fluorescencedetection instrumentation. However, for some instrumentation, such as a fluorescent gel scanner that uses a 488nm excitationwavelength, there will be minimal interference since it is the yellow dye that absorbs at this wavelength. Gels scanned by thismethod will have a light grey dye front (corresponding to the yellow dye front) below the primers.PromegaGoTaq® Green Master Mix, 2X: GoTaq® DNA Polymerase is supplied in 2X Green GoTaq® Reaction Buffer (pH 8.5),400µM DATP, 400µM dGTP, 400pM DCTP, 400µM DTTP and 3mM MgCl2. Green GoTaq® Reaction Buffer is a proprietarybuffer containing a compound that increases sample density, and yellow and blue dyes, which function as loading dyeswhen reaction products are analyzed by agarose gel electrophoresis. The blue dye migrates at the same rate as 3-5kb DNAfragments, and the yellow dye migrates at a rate faster than primers (<50bp), in a 1% agarose gel.Promega Corporation2800 Woods Hollow RoadMadison, WI 53711-5399TelephoneUSA608-274-4330Toll FreeFaxStorage Conditions: See the Product Information Label for storage recommendations.800-356-9526608-277-2516Internetwww.promega.comQuality Control AssaysPRODUCT USE LIMITATIONS, WARRANTY DISCLAIMERFunctional Assay: GoTaq® Green Master Mix is tested for performance in the polymerase chain reaction (PCR).GoTaq® Green Master Mix, 1X, is used to amplify a 360bp region of the a-1-antitrypsin gene from 100 moleculesof human genomic DNA. The resulting PCR product is visualized on an ethidium bromide-stained agarose gel.Nuclease Assays: No contaminating endonuclease or exonuclease activity detected.Promega manufactures products for a number ofintended uses. Please refer to the product label for theintended use statements for specific products.Promega products contain chemicals which may beharmful if misused. Due care should be exercised withall Promega products to prevent direct human contactEach Promega product is shipped with documentationstating specifications and othier technical information.

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PCR(polymerase chain reaction) is a molecular method to make multiple copies(amplify) of a small…

If you wanted to prepare only one PCR reaction, how much of each reagent would you add to the PCR tube?] What is the final volume of the individual PCR reactions we are making?