Pcr buffer

Genotyping of the 298bp HCG26 AluY insertion was performed by amplifying the surrounding region using primers flanking the insertion site (S1 Table). PCR was performed using 1X PCR buffer (Quanta Biosciences Inc.), 1.0mM MgCl₂, 200uM dNTPs, 500nM forward and reverse primers, 1U AccuStart II Taq polymerase (Quanta), and 100ng of DNA. Products were amplified on a 2720 thermal cycler (Applied Biosystems) at 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds. Alleles containing the insertion yielded a 1697bp product, while alleles without the insertion yielded a 1399bp product, which were resolved on a 1.5% agarose gel. Genotyping of the IL22 CNV was performed using an inventoried Taqman Copy Number Assay (Hs00146600_cn), Taqman RNAseP Copy Number Reference Assay, and 1X Taqman Genotyping Master Mix (Life Technologies) following the manufacturer’s instructions and cycling conditions. Assays were performed in quadruplicate on a 7900HT system and analyzed using CopyCaller v2.1 software (Life Technologies).

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.