Amplitaq gold 360 dna polymerase

Repaired products resulting from BER reconstituted with MEF cell extracts or purified enzymes in the context of (GAA)₂₀ and (CAG)₂₀ repeats were amplified by PCR with a forward primer (5′-CGA GTC ATC TAG CAT CCG TA-3′) and a reverse primer tagged by a 6-carboxyfluorescein (6-FAM) (5′-6-FAM-CA ATG AGT AAG TCT ACG TA-3′). PCR amplification was performed under the following conditions: 95°C for 10 min, 1 cycle; 95°C for 30 s, 50°C for 30 s and 72°C for 1.5 min, 35 cycles; 72°C for 1 h. The 6-FAM-labeled PCR products were then subjected to capillary electrophoresis using an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) (Florida International University DNA Sequencing Core Facility). The size of repaired products was determined by DNA fragment analysis with GeneMapper version 5.0 software (Applied Biosystems, Foster City, CA). Size standards, MapMarker 1000 (Bioventures, Murfreesboro, TN) were run in parallel with PCR-amplified repaired products. For all the experiments, only repaired strands were able to be amplified by PCR with AmpliTaq Gold 360 DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) used in our experiments.

RNA was extracted from transfected cells using ISOLATE II RNA Mini Kit (Bioline, Eveleigh, NSW, Australia) as per the manufacturer's instructions. The mouse dystrophin transcripts were amplified by nested-RT-PCR using SuperScript™ III Reverse Transcriptase and AmpliTaq Gold® 360 DNA Polymerase (Thermo Fisher Scientific) across exons 20–26 as described previously.^{32 (link)} The amount of RNA used for the primary amplification was 50 ng μL⁻¹ using primer set Ex20Fo (5′-CAGAATTCTGCCAATTGCTGAG-3′) and Ex26Ro (5′-TTCTTCAGCTTGTGTCATCC-3′); PCR conditions are 55 °C for 30 min, 94 °C for 2 min before entering 31 cycles of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 90 s. Then, 1 μL of this primary PCR products was subjected to the secondary PCR using primer set Ex20Fi (5′-CCCAGTCTACCACCCTATCAGAGC-3′) and Ex26Ri (5′-CCTGCCTTTAAGGCTTCCTT-3′); PCR conditions are 94 °C for 6 min before entering 33 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 2 min. The secondary PCR products were separated on 2% agarose gels in Tris–acetate–EDTA buffer and the images were captured on a Fusion Fx gel documentation system (Vilber Lourmat, Marne-la-Vallee, France). Densitometry was performed by Image J software.^{34 (link)} The actual exon-skipping efficiency was determined by expressing the amount of exon skipped RT-PCR products as a percentage of total dystrophin transcript products.