Allprep dna rna protein micro kit

The isolation of genomic DNA and RNA was performed with the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) or AllPrep DNA/RNA/Protein Micro Kit (Qiagen). For the conventional genomic PCR, 20 ng of gDNA was amplified. For the genomic qPCR, 50 pg of DNA was amplified on a LightCycler 480 (Roche, Basel, Switzerland) with iTaq universal SYBR green supermix (Bio Rad). For the analysis of mRNA expression, cDNA was synthesized from 10 ng RNA and an RT-PCR was performed on a CFX96 Connect Real-Time PCR Detection System (Bio Rad, Hercules, CA, USA) with SsoAdvanced universal SYBR green supermix (Bio Rad). For primers, see Supplemental Tables S1 and S2.

Protein isolation was performed with the AllPrep DNA/RNA/Protein Micro Kit (Qiagen), according to the manufacturer’s specifications. In short, at the end of the isolation procedure, total protein extracts recovered from the AllPrep protocol were precipitated in acetone overnight. The pellet was air-dried, and proteins were redissolved in sample buffer. The samples were boiled and loaded onto Mini-PROTEAN TGX precast gels (Biorad). Proteins were transferred onto a PVDF membrane (Trans-Blot Turbo Transfer System, Biorad) and probed with the following antibodies: Mad2l1 (1:1000, 4636, Cell Signaling); tdTomato (1:500, 600-401-379, Rockland, Limerick, PA, USA); and H4 (1:1000, 61521, Active motif, Carlsbad, CA, USA). HRP labelled Goat anti-Mouse or Rabbit secondary antibodies were used to visualize protein expression using chemiluminescence substrate (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, Waltham, MA, USA) on a ChemiDoc imaging system (Biorad). The analysis and densitometry of the protein bands was performed with ImageLab (V5.0) software.