Gel doc ez gel imager

Standard enzyme concentrations for PEN reactions were 8 U/mL polymerase, 100 U/mL nickase, 31.25 nM exonuclease, and 0.4 mM dNTPs (New England Biolabs). When cell culture conditions were not needed (Figure 1b), the dynamics of PEN reactions were recorded in 20 µL solutions inside 150 µL qPCR tubes using a Qiagen Rotor-Gene qPCR machine or a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). For biological relevance, homogeneous experiments with cells were performed in well plates (see below) and their fluorescence monitored by microscopy. To calculate the fluorescence shift, the raw fluorescence intensity was normalized by an early time point (t = 5 min), and subtracted from 1, as done previously. 35 The onset amplification time, τ , was defined as the time point at which 50% of the fluorescence of the steady state of the ON state was reached.
Polyacrylamide denaturing gel electrophoresis at 20% was run for 2 h at 200 V in 0.5X TBE buffer, stained with 1000x Sybr Gold (ThermoFisher: S11494) for 10 min, and imaged using a Gel Doc TM EZ Gel Imager (Bio-Rad). Note that we use species A 1 because upon the hydrolysis of a phosphodiester bond during the nicking event, the phosphate group remains in the 5' of the second trigger, since if the phosphate group remained on the 3' of the first trigger no autocatalytic behaviour would be attainable.

Purified UN2A and M1M2 proteins were assayed for phosphorylation by PKAc .
Phosphorylation assays used ca. 30mg of sample protein (in a 15mL volume corresponding to a protein concentration of 2mg/mL) in their final buffers. To this, 1.5mL 10x magnesium buffer (100mM MgCl2, 500mM Tris pH 7.5, 500mM NaCl) was added. Reactions contained 6.6 g/mL PKAc and were initiated by addition of ATP (at a final concentration of 1.33 mM and prepared in 50mM HEPES pH 7.5). The mixture was incubated at 37°C and 150rpm for 1h, then gently mixed overnight at room temperature. Phosphorylation was confirmed via Phos-Tag TM (Wako Pure Chemical Industries; [22] ) applied to 12% SDS-PAGE.
The SDS-PAGE gels were imaged using a Gel Doc TM EZ Gel imager and the supplied software Image Lab TM 5.2.1 (Bio-Rad). Gel densitometry was performed using Image Lab TM 5.2.1 by automatically detecting the gel bands at the default detection threshold. Relative abundance was then gathered from the lane profile generated by the software.