RD4 Subtyping was performed as developed and described by Domogalla et al. and Rettinger et al. with adaptions as following: All reactions were carried out as simplex in a total volume of 25 μl, each reaction contained 0.625 U of HotStarTaq® DNA Polymerase (Qiagen, Hilden, Germany), 0.5 μM Primer (for detailed Primer sequences see Domogalla et al.), primers flanking the 38kb deletion annealed at 60°C. After PCR amplification, the PCR products were separated by gel electrophoresis in 1% agarose gels in Tris-borate EDTA (1 x TBE) buffer at 120V for 40 min and visualized using PeqGreen from Peqlab (5μl/100ml agarose gel).
Peqgreen
Manufactured by Avantor
Sourced in Germany
PeqGREEN is a fluorescent dye used for the detection and quantification of DNA in agarose gel electrophoresis. It is a nucleic acid stain that emits green fluorescence when bound to double-stranded DNA.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin rna 2 columns, by Macherey-Nagel (2 mentions)
Gotaq polymerase, by Promega (2 mentions)
Generuler low range dna ladder, by Thermo Fisher Scientific (2 mentions)
Biodoc analyzer, by Analytik Jena (2 mentions)
Dna gel loading dye 6x, by Thermo Fisher Scientific (2 mentions)
Eos 1100d, by Canon (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
Random primer, by Promega (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase rnase h minus, by Promega (1 mentions)
Firepol master mix, by Solis BioDyne (1 mentions)
Onetaq, by New England Biolabs (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
Dnaeasy plant mini kit, by Qiagen (1 mentions)
Tae buffer, by AppliChem (1 mentions)
Tritrack dna loading dye, by Thermo Fisher Scientific (1 mentions)
Dna loading dye, by Thermo Fisher Scientific (1 mentions)
Onetaq polymerase, by New England Biolabs (1 mentions)
15 protocols using peqgreen
1
RD4 Subtyping Protocol with Adaptions
2
Semi-quantitative RT-PCR for TMEM16A and CK2 Expression
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Semi-quantitative RT-PCR was performed to detect the expression of TMEM16A and CK2 in CFBE and Cal33 cells. Total RNA was isolated using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). Total RNA (1 μg/50 μL reaction) was reverse-transcribed using random primers (Promega, Mannheim, Germany) and Reverse Transcriptase RNase H Minus (Promega, Mannheim, Germany). Each RT-PCR reaction contained sense and antisense primers for the respective gene (0.5 µM) or for GAPDH (0.5 µM), 0.5 μL cDNA and GoTaq Polymerase (Promega, Mannheim, Germany). After 2 min at 95 °C, cDNA was amplified during 30 cycles for 30 s at 95 °C, 30 s at 57 °C and 1 min at 72 °C. PCR products were visualized by loading on peqGREEN (Peqlab, VWR, Germany) containing agarose gels and analyzed using Meta Morph Version 6.2 (Molecular Devices, USA). The siRNAs—Silencer™ Select Negative Control siNEG1 (s813), siTMEM16A (HSS182856) and siCSNK2A2 (CK2α’, s3640) were purchased from ThermoFisher.
3
Reverse Transcription and Gel Electrophoresis
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Total RNA was isolated using TRIzol reagent and reverse transcribed by SuperScript III First-Strand Synthesis System according to the manufacturer's instructions (Invitrogen). Taq polymerase (Invitrogen), appropriate primers (Supplementary Tab. S1 ) and 28 cycles were used for amplification. Samples were separated on agarose gel stained with PeqGREEN (Peqlab, Erlangen, Germany). mRNA expression levels were determined relative to ACTIN expression.
4
RT-PCR for Gene Expression Analysis
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For RT-PCR total RNA from cell lines was isolated using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). Total RNA (1 µg/50 µL reaction) was reverse-transcribed using random primers and M-MLV Reverse Transcriptase RNase H Minus (all from Promega, Mannheim, Germany). Each RT-PCR reaction contained sense and antisense primers (0.5 µM) (Table 2 ), 0.5 µL cDNA, and GoTaq Polymerase (all from Promega, Mannheim, Germany). After 2 min at 95 °C, cDNA was amplified (25–35 cycles for target sequence and 25 cycles for the reference GAPDH) for 30 s at 95 °C, 30 s at 56 °C, and 1 min at 72 °C. PCR products were then visualized by loading on peqGREEN- (Peqlab, Düsseldorf, Germany) -containing agarose gels and analyzed using ImageJ.
5
Molecular Detection of Antibiotic Resistance Genes
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A c c e p t e d M a n u s c r i p t phenotypic ESBL or AmpC-production were tested for the presence of bla CTX-M (Batchelor et al., 2005) , bla SHV, bla TEM and bla OXA (Dallenne et al., 2010) . Isolates with bla CTX-M were tested for CTX-M group 1, 2 and 9 genes (Batchelor et al., 2005; Hopkins et al., 2006) . Phenotypic AmpC producers were tested for bla AmpC gene (Perez-Perez and Hanson, 2002) . All isolates were tested for the presence of qnrA, qnrB or qnr S genes (Robicsek et al., 2006) . PCR assays were performed with 5 μL of bacterial DNA, 5 pmol of each primer, 4 μL of 5x FIREPol ® Master Mix (12.5 mM MgCl2), 0.5 μl of FIREPol® DNA Polymerase 5 U/μl (Solis-Biodyne, Tartu, Estonia) and water to made up to a total reaction volume of 25 μL. PCR products were analysed by agarose gel (1.5%) electrophoresis and the DNA fragments were visualised under UV light after peqGREEN (Peqlab, Fareham, UK) staining.
6
Quantitative gene expression analysis in moss
7
Genetic Screening of Wheat Cultivars
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All cultivars were screened for known vernalization (VNR1, VRN2, and VRN3) and photoperiod (PPD1) genes. The genotyping included the recessive and dominant alleles of VRN-A1(vrn-A1, Vrn-A1a, Vrn-A1b, Vrn-A1c) (Yan et al. 2004a (link)), VRN-B1 (vrn-B1, Vrn-B1), null alleles ZCCT-A1, ZCCT-B1, and ZCCT-D1 (Zhu et al. 2011 (link)), and functional alleles ZCCT-A2, ZCCT-B2 and ZCCT-D2 of VRN2 (Distelfeld et al. 2009 (link); Kippes et al. 2016 (link)), photoperiod-insensitive alleles Ppd-A1a, Ppd-B1a, Ppd-D1a and sensitive alleles Ppd-A1b, Ppd-B1b and Ppd-D1b of Ppd1 (Beales et al. 2007 (link); Nishida et al. 2013 (link)). The primers and the protocols used to amplify the target fragments are summarized in Table S2. DNA extraction was conducted following the protocol of DNAeasy Plant Mini Kit (Qiagen, Hilden, Germany). The polymerase chain reactions (PCR) were performed in a 25 μL reaction volume containing 100 ng of genomic DNA, 1 × Taq DNA polymerase reaction buffer, 10 μM of each forward and reverse primer, 0.2 mM of dNTP, and 0.5 unit of Taq DNA polymerase (NEB, Frankfurt, Germany). The PCRs were conducted in the thermocycler Flex cycler (Analytik GmbH, Jena, Germany). PCR profiles were visualized by electrophoresis on a 1% agarose gel stained with 0.04 μl/mL peqGreen (VWR, Darmstadt, Germany).
8
Gel Electrophoresis for PCR Amplification
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Following the PCR, samples were tested for amplification success by gel electrophoresis using a 2% agarose gel (Sigma-Aldrich; A6877) in TAE buffer (AppliChem GmbH; A1691) at 4 °C. With a volume of 100 ml, the gel contained 4 µL of the dye peqGreen (VWR International GmbH; 37–500). For the loading of the agarose gel, 5 µL of the unpurified PCR product was mixed with 1 µL of 6× DNA Loading Dye (Thermo Fisher Scientific Inc.; R0611). The molecular weight marker used was composed of 1 µL Gene Ruler Low Range DNA Ladder (Thermo Fisher Scientific Inc.; SM1191), 1 µL 6 × TriTrack DNA Loading Dye (Thermo Fisher Scientific Inc.; R1161) and 4 µL nuclease-free water. Electrophoresis ran at a constant voltage of 100 V for 90–120 min (depending on the volume of the gel). The gel electrophoresis results were documented using the Quantum CX5 gel documentation system from Vilber Lourmat Deutschland GmbH (Eberhardzell, Germany) and the related BioVision software.
9
PCR Amplification and Sanger Sequencing
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Polymerase chain reaction was carried out with OneTaq polymerase (NEB) following the manufacturers’ protocol. Annealing was carried out between 55°C and at 59°C and elongation time was adjusted to the longest fragment chosen (95 s). For primer sequences see Supplementary Tables S7, S8 . 5 μl PCR product, 2.5 μl of the forward primer (10 μM) and 2.5 μl water were Sanger sequenced (Macrogen, Germany) (Supplementary Table S9 and Supplementary File 6 ). PCR products and all subsequent fragment analyses were visualized via gel electrophoresis (0.7% agarose, Roth, Germany) using peqGREEN (VWR, Germany) as dye. The 1 kbp size standard was purchased from NEB.
10
DNA Origami Folding and Purification
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The DNA origami structures were folded with 10-fold excess of oligonucleotide strands and a 100-fold excess of pyrene-modified oligonucleotides in comparison to the scaffold in 1× FOB buffer. Details of the folding program are found in ref. 33 (link) After folding, 1× Blue Juice gel loading buffer was added to the folded DNA origami which was then purified via agarose-gel electrophoresis with 1.5% agarose gel in 50 mL of FOB buffer at 80 V for 1.5 h with 2 µL peqGREEN (ordered from VWR) per 100 µL buffer. The specific band for the nanostructure was extracted from the gel. Before putting the purified DNA origami solution onto graphene, the concentration was adjusted with FOB buffer to 75 pM.
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