The in vitro selection related DNA samples and the beacon DNA were purchased from Integrated DNA Technologies (IDT, Coralville, IA). See Supplementary Table S1 for their names, sequences and modifications. For characterization, the enzyme strands were from Eurofins (Huntsville, AL). The Ln3+ other metal salts were from Sigma–Aldrich at the highest possible purity. Tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, sodium chloride and ammonium acetate were from Mandel Scientific Inc. (Guelph, Ontario, Canada). SsoFast EvaGreen supermix was from Bio-Rad for real-time polymerase chain reaction (PCR). T4-DNA ligase, deoxynucleotide (dNTP) mix, Taq DNA polymerase with ThermoPol buffer and low molecular weight DNA ladder were from New England Biolabs.
Taq dna polymerase with thermopol buffer
Manufactured by New England Biolabs
Sourced in Germany
Taq DNA polymerase with ThermoPol buffer is a thermostable DNA polymerase enzyme used for DNA amplification and synthesis. The ThermoPol buffer is optimized for use with the Taq DNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Low molecular weight dna ladder, by New England Biolabs (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Ethidium bromide, by Carl Roth (1 mentions)
Uvsolo ts, by Analytik Jena (1 mentions)
Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Dithiothreitol (dtt), by Promega (1 mentions)
Rnase free dnase, by New England Biolabs (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
4 protocols using taq dna polymerase with thermopol buffer
1
Selective DNA Amplification with Ln3+ Ions
2
PCR Amplification of Transposon Insertions
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Bacterial lysates were used as genomic DNA template for PCR. Templates were prepared by inoculating 100 µL nuclease-free water with bacterial colonies, boiling the samples in the microwave for 1 min and then performing one freeze-thaw cycle. One microliter of cell lysate was used as template for a 25-µL PCR reaction using Taq DNA Polymerase with ThermoPol Buffer (NEB). For each mutant, one primer annealed to the transposon sequence and one primer annealed to the genomic region adjacent to the predicted transposon insertion site (Table S1 ). PCR amplification was confirmed on a 1% agarose gel.
3
Analysis of mRNA Expression
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For the analysis of mRNA expression, RNA was isolated using the NucleoSpin RNA Kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s guidelines. cDNA synthesis was performed using 1 µg RNA and the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Bremen, Germany). The quantification of RNA was performed using the Primers listed below (Table 2 ) and the Platinum SYBR Green qPCR Super-Mix UDG (Invitrogen, Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer’s guidelines and assayed with a Rotor Gene 600 (Qiagen, Hilden, Germany). Reverse transcriptase PCR was performed using the Taq DNA Polymerase with ThermoPol® Buffer (New England Biolabs, Frankfurt am Main, Germany) and the primers listed below according to the manufacturer’s guidelines. Amplified RT-PCR products were separated on an 2% agarose gel (Sigma-Aldrich, Munich, Germany) with 0.001% ethidium bromide (Carl Roth GmbH, Karlsruhe, Germany) run at 100 V, and were imaged with a trans-illuminator (UVsolo TS; Biometra, Göttingen, Germany).
4
Generating AruRGP Precursor RNA Probes
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A pBluescript SKII (+) vector containing the cloned and sequenced AruRGP precursor cDNA was used to synthesize RNA probes. First, a routine PCR was performed using Taq DNA polymerase (Taq DNA Polymerase with Thermopol Buffer, New England Biolabs) and standard M13 primers (Forward: 5′‐GTAAAACGACGGCCAGTG‐3′, Reverse: 5′‐GGAAACAGCTATGACCATG‐3′, custom‐synthesized by Sigma‐Aldrich) to linearize the plasmid and amplify the insert. The PCR product, which included the AruRGP precursor cDNA sequence and T3 and T7 RNA polymerase sites, was purified using a QIAquick gel extraction kit (Qiagen).
RNA probes were synthesized from the PCR product using a digoxygenin (DIG)‐labeled nucleotide triphosphate mix (Roche, Nutley, NJ) supplemented with dithiothreitol (Promega), a placental RNase inhibitor (Promega), and RNA polymerases (New England Biolabs), according to the manufacturer's instructions. T3 or T7 RNA polymerase was used for synthesis of the antisense or sense probes, respectively. Reaction products were digested with RNase free DNase (New England Biolabs) to remove template DNA and then stored at –20°C in 25% formamide made up in 2× saline‐sodium citrate (SSC) buffer.
RNA probes were synthesized from the PCR product using a digoxygenin (DIG)‐labeled nucleotide triphosphate mix (Roche, Nutley, NJ) supplemented with dithiothreitol (Promega), a placental RNase inhibitor (Promega), and RNA polymerases (New England Biolabs), according to the manufacturer's instructions. T3 or T7 RNA polymerase was used for synthesis of the antisense or sense probes, respectively. Reaction products were digested with RNase free DNase (New England Biolabs) to remove template DNA and then stored at –20°C in 25% formamide made up in 2× saline‐sodium citrate (SSC) buffer.
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