Faststart taq polymerase

Human reference mRNA sequences for 263 genes were retrieved from the NCBI Reference Sequence Database and individually aligned to their respective Pongo abelli (Sumatran orangutan) genome sequence (NCBI Pongo_pygmaeus_abelii-2.0.2 assembly) via BLAST to find homologous sequence stretches containing as few nucleotide ISVs as possible. The genes in this study were chosen based on our own research interests: the majority of the genes are related to epigenetic regulation mechanisms (Supplementary Table 3). Homologous sequence stretches were subjected to Primer322 (link) to design primer pairs that bind both human and orangutan sequences simultaneously in PCR amplicon while possessing the small numbers of ISV nucleotides in its amplicons to discriminate species origin. Primers were pooled into 24 groups of ~20 pairs in such a way that multiple primer pairs for a single transcript are separately assigned to different groups, to avoid the production of unintended amplicons. A multiplexed PCR reaction was carried out using each primer group and either of two Taq polymerases, Solg^TM h-Taq DNA polymerase (SolGent, Korea) or FastStart Taq polymerase (Roche) for the Rep1 set or Rep2 set, respectively, with the following conditions: 15 min of enzyme activation at 95 °C followed by 40 cycles of 95 °C for 20 s, 55 °C for 25 s, and 65 °C for 2 min.

cDNA constructs of the human GLP-1 receptor containing a myc-tag was donated by Professor Patrick Sexton (Monash University, Australia). cDNA for human GLP-1 receptors was provided by Dr Graeme Wilkinson (AstraZenica, UK). cDNA for the glucagon receptor (GCGR) was purchased from the Missouri University of Science and Technology cDNA Resource Centre (http://cdna.org). DNA manipulations were performed using standard methods. Oligonucleotides were supplied by Invitrogen. PCR amplification used FastStart Taq polymerase (Roche Diagnostics, Burgess Hill, UK). All constructs were sequenced by GATC (GATC Biotech, London, UK) prior to use.

RNA was reverse transcribed to cDNA using the Transcriptor First-Strand cDNA Synthesis Kit (Life Technologies) with oligo dT primers. Primers specific for the YWHAE-NUTM2 fusion transcript derived from t(10;17)(q22;p13), were taken from O'Meara et al., 2012 [7 (link)]. Amplification was performed for 40 cycles with FastStart Taq Polymerase (Roche) and electrophorectic bands were visualized on ethidium bromide stained gels. Validation of BCOR ITD was performed on tumor DNA and cDNA with primers located on exon 15, outside the duplicated region (Fw 5′-CCATTGCAGAGGCAGAATTTTA-3′ and Rev 5′-CTGTACATGGTGGGTCCAGCT-3′). PCR products were cutted and purified from gel and sequenced using the Big Dye Terminator v1.1 cycle sequencing kit (Life Technologies) on ABI Prism 3730.
Gene expression comparison between 8 CCSK cDNA and a control group of other renal tumors (5 Wilms tumor samples) was performed by quantitative RT-PCR, with primers located on exons 12 and 13 (Fw 5′-CTCTTATGGTGCTGACCCCACC-3′; Rev 5′-CCACTGGCGTCATCATCATTG-3′).

DNA isolation from blood, liver tissue (OI calf) and semen samples was performed using MagNa Pure LC DNA Isolation Kit I (Roche Diagnostics, Mannheim, Germany) or a modified salting out procedure (Miller et al. 1988 (link)).
Using NCBI/Primer-BLAST, PCR primer pairs were designed (supplementary materialTable 1) (Ye et al. 2012 ). A total volume of 25 μl was prepared for each PCR reaction, including 1× PCR reaction buffer plus 20 mM MgCl₂, 0.5 μl of 10 mM dNTPs, 1 U FastStart Taq Polymerase (Roche), 1 μl of 10 pmol/μl each primer (Sigma-Aldrich, Hamburg, Germany), and 1 μl of 20 ng/μl DNA. Reaction conditions included 95 °C for 10 min, then 30 cycles of 95 °C for 30 s, primer-specific annealing temperatures (supplementary material Table 1) for 30 s and 72 °C for 45 s, followed by final elongation at 72 °C for 7 min.
MOVE TABLE 1 to paragraph 3.1.
Purification of PCR products was performed using Rapid PCR Cleanup Enzyme Set (New England Biolabs GmbH, Frankfurt am Main, Germany). Amplicons were sequenced using the BigDye^TM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific GmbH, Schwerte, Germany) and fragments were separated on an ABI PRISM 3130xl Genetic Analyzer (Life Technologies, Darmstadt, Germany) following the manufacturers´ protocols. DNA sequences were aligned using SeqMan Pro software (DNASTAR Lasergene, Madison, WI, USA).

The plasmid for the generation of direct, in-frame mCherry fusion proteins was kindly donated by Steve Royale (University of Warwick). cDNA constructs of the human GLP-1R (containing an N-terminal Myc tag) and the human glucagon receptor were donated by Professor Patrick Sexton (Monash University, Australia) and Dr. Run Yu (Geffen School of Medicine at UCLA, Los Angeles, CA), respectively. Constructs for the expression of N-terminally FLAG-tagged human RAMPs were described previously (10 (link)). The cDNA construct containing a Myc-tagged CLR was provided by Dr. Michel Bouvier (University of Montreal, Canada). RAMP-GFP constructs were purchased from Cambridge Bioscience (Cambridge Bioscience Ltd., Cambridge, UK). DNA manipulations were performed using standard methods. Oligonucleotides were supplied by Invitrogen. PCR amplification used FastStart Taq polymerase (Roche Diagnostics, Burgess Hill, UK). All constructs were sequenced by GATC (GATC Biotech, London, UK) prior to use.

Single marker PCRs for detection of 8 specific Mse fragments for the development of QC1 assay were conducted as previously described (Schardt et al, 2005 (link)). For all PCRs, 0.5 μl WGA product was used as template; oligonucleotide sequences and corresponding annealing temperature T_A are listed in Supplementary Table S2. Single cell metaphase CGH was done according to Klein et al (2002 (link), 1999 (link)) to validate the selected primer pairs for the QC1 assay.
For the multiplex PCR (QC2 assay), 1 μl WGA template was used in 10 μl of a water-based mastermix containing 1× FastStart PCR Buffer (including MgCl₂), 200 nM dNTPs, 0.5 U FastStart Taq Polymerase and 4 μg BSA (all consumables Roche Diagnostics GmbH, Germany). The eight primers of QC2 assay (KRAS, D5S2117, TP 53 Exon 2/3 and CK19, see Supplementary Table S2) were each used in an end concentration of 0.4 μM. PCR was started with a first step at 95°C for 4 min, followed by 32 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 90 s, and a final elongation step of 7 min at 72°C. To determine the genome integrity index, PCR products were visualized on a 1.5% agarose gel. The protocol of the multiplex PCR assay is the basis for the now commercially available Ampli1™ QC kit (Silicon Biosystems spa).