In 1993, Applied Biosystems was acquired by Perkin-Elmer, the old-line scientific instrument company in Norwalk, Conn., that had rights to the polymerase technology.
Other available methodologies that allow generation of ChIP-seq data from limited amounts of input material rely on an extra amplification step, based either on custom designed random primers or T7 RNA-polymerase technology [ 5, 6].
This PCR contained 1.25 U Taq DNA polymerase (Life technologies, Rockville, MD) in 1x PCR buffer (Life technologies, Rockville, MD), 0.2 mM of each dNTP, 2.5 mM MgCl2, and 1 μM of each primer.
This PCR reaction contained 1.25 U Taq DNA polymerase (Life technologies, Rockville, MD) in 1x PCR buffer (Life technologies, Rockville, MD), 0.2 mM of each dNTP, 2.5 mM MgCl2, and 1 μM of each primer.
The PCR conditions were 1× PfuTurbo Cx reaction buffer, 0.2 mM of each dNTP, 0.5 µM of forward and reverse primers, 1 U of PfuTurbo Cx hotstart DNA polymerase (Agilent Technologies, California, USA) and 60 ng of converted DNA.
Genomic DNA was extracted (Harju et al. 2004) from three colonies in each strain and amplified by PCR (C1000 Touch™ thermocycler, Bio-rad, USA) using Dream taq polymerase (Life technologies, Sweden) and selected primers targeting (1) TY3, TY3 elements (Transposable elements, individually and combined) (i Nogué et al. 2012; Schofield et al. 1995).
The coding sequence for Hfq was PCR amplified from F. novicida using PfuUltra II Fusion HotStart Polymerase (Agilent Technologies) and primers FN-hfq-ATG-F and FN-hfq-330R-PstI (Table 2) at 50°C annealing.
RT genes in viral cDNA were amplified by PCR, using Pfu Ultra II Hotstart Polymerase (Agilent Technologies, Santa Clara, CA) and primers RT amp 5' (5' upstream of HIV RT, within SIV pol, 5-TACTAAAGAATACAAAAATGTAGA-3), and RT amp 3' (3' downstream of RT 5-CTCTGTGGATTGTATGGTACCCC-3).
To create REST promoter regions in which mutations were introduced in the putative NF-kappa B binding sites, deletion constructs A, B and C were amplified by PCR using Pfu DNA polymerase (Agilent Technologies) and sets of primers, which contained mutated NF-kappa B binding sites (supplemental Table S2).
Similarly, the N62 pool consisted of a 62-nucleotide (N62) region flanked by a different pair of primers: 5' - GATAATACGACTCACTATAGGCGCTCCGACCTTAGTCTCTG-N62-GAACCGTGTAGCACAGCAGA - 3.' Following in vitro transcription by T7 RNA polymerase (Epicenter Technologies, Madison, WI), the derived RNA library was purified on an 8% denaturing polyacrylamide gel, then eluted and precipitated.
PfuTurbo C x Hotstart DNA polymerase (Agilent Technologies, Böblingen, Germany) was used for high fidelity PCR.
