Difference between revisions of "Part:BBa K4344026"

Latest revision as of 14:42, 12 October 2022

HSV-UL19-rev-1

Part of our Sequencing primer set used for verification of various cloning steps throughout our project. Part of primer sets for qPCR of UL19 copies together with following parts: BBa_K4344021 (HSV-UL19-fwd-2), BBa_K4344022 (HSV-UL19-fwd-3), BBa_K4344023 (HSV-UL19-fwd-1), BBa_K4344024 (HSV-UL19-rev-2), BBa_K4344025 (HSV-UL19-rev-3), BBa_K4344026 (HSV-UL19-rev.-1)

Usage and Biology

Three different Primer sets were evaluated concerning their capabilities to quantify eGFP or UL19 enscripts. For this purpose Primers were first evaluated in a temperature gradient PCR since the used kit needs two different annealing temperatures, one for reverse transcription and one for amplification for the target gene by conventional PCR. The used gradient ranged from 53.8 °C to 64 °C.

Furthermore all three primers were evaluated for each target in a plasmid titration assay. Aim of the assay is to determine cut of values for Kit and primer per combination and determine primer efficiency. Titration curves for each primer set and target are shown in Figure 6. Figures 6G and 6H show the calculated primer efficiency based on the obtained slope of the titration regression model.

For the UL19, primer set 1 shows a R² of 0.9956 and an efficiency of 104.91%. Primer set 2 shows a R² of 0.9995 and an efficiency of 99.74%. Primer set 3 a R² 0.9998 and efficiency of 100.20%. Primer set 2 and 3 show overfitting and high efficiency, while primer set 1 shows no overfitting at very high efficiency. A RT-qPCR or qPCR for UL19 transcripts derived from pRK5-HSV UL19 plasmid should be carried out with primer set 1.

Figure 6: Titration curves and primer efficiency values All titration curves range from 10^10 to 10^2 copy per well. A: Titration curve of eGFP Primer Set 1. B: Titration curve of UL19 Primer Set 1 C: Titration curve of eGFP Primer Set 2 D: Titration curve of UL19 Primer Set 2 E: Titration curve of eGFP Primer Set 3 F: Titration curve of UL19 Primer Set 3 G: Overview of calculated primer efficiency of eGFP Primer sets. H: Overview of calculated primer efficiency UL19 Primer sets.

Sequence and Features