Accustart 2 geltrack pcr supermix

Genomic DNA and total RNA were extracted from bladder cell lines and Cohort 2 using AllPrep DNA/RNA Kit (Qiagen, Germantown, MD, USA) and cohort 3 using QIAamp DNA FFPE Tissue Kit (Qiagen). Extracted DNA from Cohort 1 was provided by EDRN. PCR primers were designed to amplify the promoter and each exon in PAI1. DNA were used for PCR amplification. PCR reactions were carried out in a total volume of 25 µL containing genomic DNA template, 0.4 µM of each PCR primer, and GoTaq G2 Hot Start Green Master Mix (Promega, Madison, WI, USA), except for PAI1 exon 9. Due to the length of exon 9′s PCR products, AccuStart II GelTrack PCR Super Mix (Quanta BioSciences, Inc., Gaithersburg, MD, USA) was used. Forty cycles of 30 s at 94 °C, 30 s at 60 °C, and 30–120 s at 72 °C (per intended PCR product) were performed in a programmable thermal cycler (Bio-Rad, Hercules, CA, USA). PCR products were checked on a 1.5% agarose gel, followed by PCR purification and bidirectional Sanger sequencing (Psomagen, Rockville, MD, USA). Sequence analysis was performed using Geneious, (Geneious version 8, “http://www.geneious.com (accessed on 5 August, 2015)”, [51 (link)]) and detected variants were confirmed against a RefSeq genomic DNA sequence (PAI1: NG_013213.1). All genetic alterations identified by Geneious were compared to the NCBI SNP database (dbSNP).

Genomic DNA was extracted using the Extracta DNA Prep for PCR kit and genotyped with AccuStart II GelTrack PCR SuperMix (Quanta Biosciences, Gaithersburg, MD, USA). Primers used to genotype and sex animals are available in Supplementary Table 1. PCR was run in a 2720 ThermoCycler (Applied Biosystems, Waltham, MA, USA) with the following program: 94° for 3 minutes; (94° for 30 seconds, 60° for 30 seconds, 72° for 1 minute) x 30 cycles; 72° for 10 minutes.

Genotyping was performed as described previously [49 (link)]. Genotyping samples were collected at weaning and/or during dissection. Ear or tail samples were routinely collected for DNA analysis. Extracta DNA Prep for PCR-tissue (Quanta Biosciences, Gaithersburg, MD, USA) was used to isolate genomic DNA from each mouse. Genotyping of each animal was performed using AccuStart II GelTrack PCR SuperMix (Quanta Biosciences, USA). After PCR, samples were analyzed on a 2% agarose gel by gel electrophoresis in tris-acetic acid-EDTA (TAE) buffer (Elatus Media Kitchen, University of Helsinki). Genotyping primers used are summarized in Table 1.