Recombinant human IL8 was serially diluted in PLA buffer (1 mM D-biotin (Life Technologies), 0.1% BSA (Sigma-Aldrich), 0.05% Tween 20, 100 nM goat IgG (Sigma-Aldrich), 0.1 µg/µl salmon sperm DNA (Life Technologies), 5 mM EDTA in PBS), or in either 10% or 50% chicken serum prepared in PLA buffer. Individual dilution series included a negative control with no spiked antigen. Two µl of each sample were mixed with 2 µl of 60 pM PLA probes (either purified anti-IL8-Arm1_long and anti-IL8-Arm2_long conjugates, unpurified anti-IL8-Arm1 and anti-IL8-Arm2 conjugates, or a pair of purified conjugates with spiked-in anti-IL8 or Arm1 and Arm2, respectively) and incubated for 1.5 h at RT. After incubation, 1 µl of the mixture was transferred to 25 µl ligation and quantitative PCR mixture (1× PCR buffer (Quanta Biosciences), 2.5 mM MgCl2 (Quanta Biosciences), 0.5× Sybr Green I (Life Technologies), 0.1 µM BioFwd primer, 0.1 µM BioRev primer, 0.025 µM BioSplint, 0.08 mM ATP (Thermo Scientific), 0.2 mM dNTPs (with dUTP) (Thermo Scientific), 0.03 U/µl AccuStart Taq DNA polymerase (Quanta Biosciences), 0.01 U/µl T4 DNA ligase (Thermo Scientific) and 0.002 U/µl Uracil N-glycosylase (Thermo Scientific)). Quantitative PCR was performed in an MX3005 cycler (Stratagene) with an initial incubation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
Accustart taq dna polymerase
Manufactured by Quanta Biosciences
Sourced in United States
AccuStart Taq DNA polymerase is a thermostable DNA polymerase enzyme used for DNA amplification in polymerase chain reaction (PCR) techniques. It is capable of synthesizing new DNA strands complementary to a template DNA sequence.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
D biotin, by Thermo Fisher Scientific (1 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (1 mentions)
Uracil n glycosylase, by Thermo Fisher Scientific (1 mentions)
Pcr buffer, by Quanta Biosciences (1 mentions)
Ion pgm template ot2 200 kit, by Thermo Fisher Scientific (1 mentions)
Ion pgm sequencing 200 kit v2, by Thermo Fisher Scientific (1 mentions)
Tbe gel, by Thermo Fisher Scientific (1 mentions)
4 protocols using accustart taq dna polymerase
1
Quantitative Protein Detection by PLA
2
Targeted DNA Sequencing of L265P Mutation
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PCR primers were designed (F: 5’-ggttgaagactgggcttgtc-3’, R: 5’-gcgagtccagaaccaagatt-3’) using Primer 3 Plus to amplify an amplicon of 198bp spanning the L265P on genomic DNA. 30ng of DNA were amplified by PCR using AccuStartTaq DNA Polymerase (Quanta BioSciences) according to manufacturer’s instructions. Library preparation for Ion Torrent was performed using the NEBNext Fast DNA Library Preparation Kit (New England Biolabs), template preparation was done on the Ion OneTouch2 instrument using the Ion PGM Template OT2 200 Kit, followed by sequencing on an Ion Torrent PGM sequencer using the Ion PGM Sequencing 200 Kit v2 (Life Technologies).
3
HPV Detection by PCR and RT-PCR Amplification
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HPV-DNA detection was performed by PCR amplifying 50 ng per sample using the primers GP5+/GP6+, as described previously [28] (link). For all (RT)-PCR reactions the Quanta Biosciences Perfecta PCR mix, utilizing the AccuStart™ Taq DNA-Polymerase, was used. PCR conditions were as follows: initial denaturation: 15 min 95 °C, followed by 40 cycles of 1 min 95 °C, 1 min 40 °C and 1 min 72 °C, and a final elongation: 5 min 72 °C. DNA integrity was analyzed using genomic primers for the housekeeping gene ß2-microglobin (B2M; Promolgene) according to the manufacturer's protocol; in brief, initial denaturation 10 min 95 °C; followed by 40 cycles:20 s 95 °C, 20 s 60 °C and 20 s 72 °C. Additionally, a positive control (a synthetic oligonucleotide of the HPV L1 gene, covered by the GP5+/GP6+ primers; Eurofins; Ebersberg Germany) was amplified in the GP5+/GP6+ PCRs. Amplification products were sequenced by Sanger sequencing and alignments were obtained from the GenBank online BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). HPV-RNA detection was performed by RT-PCR, using 10 ng cDNA, generated as described above, and HPV-type specific E6/E7 primers [29] (link) under following PCR conditions: initial denaturation: 10 min 95 °C followed by 40 cycles:15 s 95 °C, 45 s 60 °C.
4
Genotyping of Methionine/Valine Polymorphism
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A 113 bp fragment was amplified using a forward 5′-GAGGCTTGACATCATTGGCT and reverse primer 5′-CGTGTACAAGTCTGCGTCCT [48 (link)]. The 25 μL amplification mixture contained 50 ng of genomic DNA, 0.2 μM of each primer and 1X AccuStart Taq DNA polymerase (Quanta Biosciences, Gaithersburg, MD, USA). The cycling conditions were: a) initial denaturation at 95 °C for 2 min; b) 35 cycles at 94 °C for 30 s; c) 60 °C for 30 s; d) 72 °C for 30 s; e) final extension following the completion of the cycles at 72 °C for 5 min [49 (link)]. The 7.5 μL PCR product was cut with the restriction enzyme Eco 721 (Fermentas, Glen Burnie, MD, USA) for 3 h at 37 °C. The bands were separated using a 4-20 % TBE gel (Invitrogen, Carlsbad, CA, USA) at 14 mA for 60 min. The resulting banding pattern for the methionine and valine allele was 113 bp and two bands at 78 and 35 bp, respectively.
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