Difference between revisions of "Part:BBa K4344010"

Revision as of 14:35, 12 October 2022

EGFP-fwd-2

Part of our Sequencing primer set used for verification of various cloning steps throughout our project. Part of primer sets for qPCR of EGFP copies together with following parts: BBa_K4344009 (EGFP-fwd-1), BBa_K4344010 (EGFP-fwd-2), BBa_K4344011 (EGFP-fwd-3), BBa_K4344012 (EGFP-rev-1), BBa_K4344013 (EGFP-rev-2), BBa_K4344014 (EGFP-rev-3).

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.

The evaluation of our qPCR primers shows that three primer sets for both targets are functional at the proposed temperatures for reverse transcription 55°C, as well as for amplification by PCR at 60°C. Titration curves for eGFP primer set 1 and 2 show overfitting for the target due to their R² of 0.9994 and 0.999 for primer set 1 and 2 respectively. Primer set 3 shows a R² of 0.9988 and therefore no over- or underfitting. In case of primer efficiency, primer setb1 shows an efficiency of 96.18%, primer set 2 of 96.86% and primer set 3 of 80.84%. Therefore considering the overfitting and calculated primer efficiency, RT-qPCR or qPCR for eGFP transcripts derived from pEGFP C1 plasmid should be carried out with primer set 2 since it has the highest efficiency and a smaller overfitting than 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