Purification: For the detection of the Cy5 molecules, incorporated during RPA, by gel electrophoresis and fluorescence spectroscopy, the RPA products were previously purified using magnetic beads. For this purpose, the Mag-Bind® Total Pure NGS Kit (Omega Bio-Tek; M1378-01) was used according to the manufacturer's instructions. The elution step was performed in 25 µl ddH2O.
Gel electrophoresis: To view the labelled and unlabelled amplicons, they were separated and analysed according to their size after RPA and appropriate purification on a 2% agarose gel in 1 × TAE buffer (50 × TAE-buffer; PanReac AppliChem; A1691). For detection of all bands and markers, 2 µl peqGreen was added to the gel (VWR; 732–3196). To 15 µl of each DNA-sample, 3 µl of DNA Gel Loading Dye (6 x) (Thermo Fisher Scientific; R0611) was added and electrophoresis was performed at 120 V for 1.5 h. Gene Ruler Low Range DNA Ladder (Thermo Fisher; SM1193) and peqGOLD DNA-ladder mix (VWR; 25–2040) came into use for the size assignment of the gel bands. For the detection of the gels the LS Reloaded Microarray Scanner (Tecan) was used. Here, a custom 3D-printed holder for the gels was used. With this modified method, both the green channel for the recognition of the DNA intercalator (excitation at 532 nm) and the red channel (excitation at 633 nm) for the incorporated Cy5 molecules could be analysed under the same conditions at the same time.
Fluorescent spectroscopy: For the determination of the incorporation rates of Cy5 molecules into the RPA products, 20 µl of each previously purified sample was analysed in FLUOstar Omega (BMG Labtech). Excitation at 620 nm and a 680 nm emission filter were used. For the calibration line needed to calculate the incorporation rates, a Cy5-dUTP dilution series in the range of 2.0–20.0 µM was measured under the same conditions.
Gel electrophoresis: To view the labelled and unlabelled amplicons, they were separated and analysed according to their size after RPA and appropriate purification on a 2% agarose gel in 1 × TAE buffer (50 × TAE-buffer; PanReac AppliChem; A1691). For detection of all bands and markers, 2 µl peqGreen was added to the gel (VWR; 732–3196). To 15 µl of each DNA-sample, 3 µl of DNA Gel Loading Dye (6 x) (Thermo Fisher Scientific; R0611) was added and electrophoresis was performed at 120 V for 1.5 h. Gene Ruler Low Range DNA Ladder (Thermo Fisher; SM1193) and peqGOLD DNA-ladder mix (VWR; 25–2040) came into use for the size assignment of the gel bands. For the detection of the gels the LS Reloaded Microarray Scanner (Tecan) was used. Here, a custom 3D-printed holder for the gels was used. With this modified method, both the green channel for the recognition of the DNA intercalator (excitation at 532 nm) and the red channel (excitation at 633 nm) for the incorporated Cy5 molecules could be analysed under the same conditions at the same time.
Fluorescent spectroscopy: For the determination of the incorporation rates of Cy5 molecules into the RPA products, 20 µl of each previously purified sample was analysed in FLUOstar Omega (BMG Labtech). Excitation at 620 nm and a 680 nm emission filter were used. For the calibration line needed to calculate the incorporation rates, a Cy5-dUTP dilution series in the range of 2.0–20.0 µM was measured under the same conditions.
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