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(agarose gel electrophorosis, real time pcr-amount of amplification…
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real time pcr-amount of amplification product is measured as pcr processes, convention is after
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thermocyclers have fluorescent detection modules to monitor signal, which increases as amt of dna product increases
initiation phase-fluorescent remains at background level, pcr product amount increases but fluorescence increase is undetectable
exponentation phase-detectable fluorescence signal-happens when fluorescence intensity passes threshold which is the cq value
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can determine initial no. of template dna, is qualitative and quantitative, data can be visualised without additional post amplification step, reduce chance of contamination and mix up
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rapid process, highly specific, sensitive and versatile
sequence needs to be known, prone to non specfic amplification due to parameters not optimised or contamination taq polymerase no proofreading ability
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avoid segments with >3 consecutive repeated nucleotides, avoid complementary sequences within and between primer
after coding the reverse primer in the 3 to 5 direction, flip it so it goes 5 to 3 from the left