Taqman Detection in Real Time PCR

G. Ronald Jenkins & Louis Bussjaeger, Deana Namuth & Leah Sandall Author

03/15/2019 Added

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Real time PCR can be accomplished using the taqman detection system. This video shows an overview of the taqman process.

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[00:00:06.280]This video clip shows an overview of setting up a real time PCR experiment
[00:00:11.580]using a Taqman probe format.
[00:00:16.800]Lewis has prepared a master mix solution and is seen here mixing it using a
[00:00:21.489]repeating pipette.
[00:00:23.480]This master mix contains primers and labeled probes specific for the gene he
[00:00:28.745]is tested for as well as nucleotides, water buffer, intact polymerase.
[00:00:34.560]With the repeating pipetter, he alloquats 20 microliters into
[00:00:38.316]individual wells of the microtiter plate.
[00:00:41.520]The repeating pipetter allows him to quickly and accurately add the reagents
[00:00:46.338]to each individual well.
[00:00:48.880]Next, he adds replicate DNA samples to the
[00:00:51.582]corresponding wells.
[00:00:53.400]Some will have standard DNA, which have known concentrations of the
[00:00:57.195]target for a gene known to be present in the plant's genome.
[00:01:01.280]These will serve as controls in his experiment.
[00:01:04.600]DNA for his experiment was isolated from his seed samples in other laboratory
[00:01:09.446]procedures prior to this.
[00:01:11.000]Taqman PCR gene amplification in individual wells is measured by the
[00:01:16.302]amount of fluorescence given off by his Taqman probe at the end of each PCR cycle.
[00:01:23.760]Other wells will contain DNA samples which will allow him to quantitatively
[00:01:28.397]measure the amount of target gene present.
[00:01:31.760]Amplification performance of his unknown samples will be compared with those of
[00:01:36.929]his known DNA standards.
[00:01:44.800]After dispensing all reaction components, Lewis covers the microtiter plate with a
[00:01:50.324]prism optical adhesive cover to seal the wells.
[00:01:57.040]This prevents cross contamination and evaporation of the samples throughout the
[00:02:01.593]heating and cooling cycles in the thermal cycler.
[00:02:08.680]Finally, he places a compression pad on top of the
[00:02:11.770]adhesive cover.
[00:02:13.120]This ensures a good seal around each well.
[00:02:17.120]Now he enters data and sets up the computer program for a 2 step PCR
[00:02:22.003]reaction.
[00:02:23.920]Next, Lewis puts the 96 well microtiter plate
[00:02:26.573]into the thermal cycler for heating and cooling.
[00:02:30.040]The computer collects fluorescence data at the end of each cycle from individual
[00:02:34.955]wells.
[00:02:35.960]By comparing the amplification efficiency of his standard DNA to those of his
[00:02:42.165]unknown samples, the DNA can be analyzed to quantify the
[00:02:46.700]amount of target gene present in this graph.
[00:02:50.360]The red horizontal line identifies ACT threshold.
[00:02:54.600]This is by definition the PCR cycle at which a samples fluorescent signal is
[00:02:59.929]first detected above a baseline value.
[00:03:03.320]This is the point where target genes are quantified,
[00:03:06.675]so the sooner a sample crosses this line, the more target gene it initially
[00:03:11.486]contained.
[00:03:12.560]The computer software package generates a standard curve of CT versus fluorescence
[00:03:18.139]signal for all standard DNA samples.
[00:03:20.920]Then by interpolation, it calculates the starting copy number
[00:03:25.136]for Lewis's unknown samples.

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Taqman Detection in Real Time PCR

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