Syber safe dna gel stain

For semi-quantitative PCR and quantitative real time PCR, total cellular RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and reverse transcription (RT) was performed with Quantitect Reverse Transcription Kit (QIAGEN). For semi-quantitative PCR, the Ready Mix Taq PCR kit (Sigma-Aldrich) was used. The oligonucleotides 5′-CCTTGTAGTTGAGAACCAGG-3′ and 5′-GGGGCTTGGTATATATGTGG-3′ (Eurofins MWG Operon, Ebersberg, Germany) were used for amplification of the XBP-1 transcript fragments. PCR products were resolved on 2% agarose gels, stained with Sybersafe DNA gel stain (Life Technologies, Darmstadt, Germany) and visualised under ultraviolet illumination using Fusion image capture (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5′-GTCGTGGATCTGACGTGCC-3′ and 5′-GATGCCTGCTTCACCACCTT-3′) was amplified as internal control. For quantitative real time PCR, QuantiTect Primers for IRE1α, BiP, ATF4, CHOP, CB1 and GAPDH were purchased from QIAGEN and run with the QuantiFast SYBR Green PCR Kit (QIAGEN) on a CFX96 Real Time PCR Detection System (BioRad, Munich, Germany). Results were analysed with the CFX Manager v2.0 and Rest 2008 software and normalised to GAPDH mRNA content for each sample.

Ectoplacental cones were treated with 50 μl proteinase K (60 μg/ml) in PBND [50 mM KCl, 10 mM tris-HCl, 2.5 mM MgCl₂, gelatin (0.1 mg/ml), 0.45% NP-40, and 0.45% Tween 20], for 1 hour at 55°C to extract DNA for PCR. To deactivate proteinase K so that it does not interfere with DNA amplification, they were then incubated at 95°C for 10 min. To genotype mouse embryos as either Nipbl^Flox/+ (WT) or Nipbl^FIN/+ (Nipbl^+/−) and ESCs as either Nipbl^Flrt/+ (WT) or Nipbl^FLEX/+ (Nipbl^+/−), standard Taq PCR was performed on the extracted DNA using the primers and thermocyling protocol previously described in (24 (link)): Primer 1, 5′-CTCCGC CTCCTCTTCCTCCATC-3′; primer 2, 5′-CCTCCCCCGTGCCTTCCTTGAC-3′; primer 3, 5′-TTTGAGGGGACGACGACAGTCT-3′. Thermocycling conditions are 1 cycle a 95°C for 30 s; 30 cycles of 95°C for 1 min, 59°C for 30 s, and 68°C for 1 min; and 1 cycle at 68°C for 5 min and hold at 4°C. PCR products were treated with loading dye and electrophoresed in agarose gel stained with SYBER Safe DNA Gel Stain (Invitrogen, S33102) without cleanup and visualized under ultraviolet light. Flox conformation is 782 base pairs (bp), FIN conformation is 518 bp, Flrt conformation is 735 bp, and FLEX conformation is 652 bp.

Bacteria were grown from glycerol stocks on Tryptone Soya Agar (TSA) (Oxoid, Basington, UK) plate overnight at 37 ± 1 °C. Genomic DNA was extracted using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany), following the manufacturer’s instruction.
A multiplex PCR targeting four genes (lacY, lacZ, uidA, cyd) was used for E. coli identification, following the method described by Horakova et al. (2008) [115 (link)].
The PCR amplification was performed in a reaction volume of 10 µL containing 5 µL REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, St Louis, MO, USA), 0.25 µL primers (10 pmol) and 1.5 µL DNA.
The following amplification parameters were applied: initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 25 s, elongation at 72 °C for 30 s and a final extension at 72 °C for 3 min.
The amplified products were loaded onto a 2% agarose gel containing Syber Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) and run in 1X TBE buffer at 100 V for 1 h.
PCR fragments were visualized with a UV transilluminator. A pUC19 DNA/MspI (Hpall) Marker (Thermo Fisher Scientific, Waltham, MA, USA) was loaded on each gel as a DNA size standard. E. coli ATCC 25,922 DNA was present in every run as a positive control strain. Strains showing PCR products of 463 bp, 393 bp, 319 bp and 264 bp were considered E. coli.