Phusion flash high fidelity dna polymerase

All inserts were PCR amplified from Jurkat genomic DNA (for miRNA constructs and 3'UTR constructs) using Phusion Flash High-Fidelity DNA Polymerase (Thermo Fisher Scientific, USA) under the following conditions: 98°C for 10 seconds, 30 cycles of: 98°C for 10 seconds, 60°C for 5 seconds, 72°C for 15 minutes, and final extension at 72°C for 10 minutes. Double digestion was performed in the presence of two restriction enzymes (New England Biolabs (NEB), USA) and their compatible buffer for 1 hour at 37°C, according to the manufacturer’s instructions. The restriction product was run on a 1.5% agarose gel, excised and then purified using Wizard SV Gel and PCR Clean-Up kit (Promega, USA) to eliminate excess nucleotides and primers. The clean product was then ligated to the complementary plasmid using T4 DNA ligase (NEB, USA) for 1 hour at 37°C and transformed into DH5α competent E. coli cells (Bio-Lab, Israel). Cells were grown overnight on 0.1% ampicillin LB-agar plates at 37°C. Colonies were selected and grown overnight in a 37°C shaker in 0.1% ampicillin liquid LB. Plasmids were extracted using the NucleoSpin midi-prep kit (Macherey-Nagel, Germany) and were Sanger-sequenced for verification.

The TissueScan Human Major Tissue Plate by OriGene Technologies (Lot#TH28) was used to determinate expression of SLC10A7 transcript variants in different human tissues. For PCR amplification, Phusion Flash High-Fidelity DNA Polymerase (Thermo Scientific), forward primer (5′-GGC TTT TAA AAG GTT TGC AGA CAG TAG G-3′), and reverse primer (5′-GAG GCA ACA TTC ACA AGT ACA AGT CTT CAG-3′) were added to the pre-spotted cDNA panel in each well. PCR amplification was performed on the Applied Biosystems 7300 Real-Time PCR System at 45 cycles under the following conditions: initialization for 90 s at 98 °C, denaturation for 30 s at 98 °C, annealing for 30 s at 64 °C, extension for 1 min at 72 °C, final elongation for 1 min at 72 °C and final hold at 4 °C. After amplification, PCR products were separated by 2.5% agarose gel electrophoresis in TAE-buffer. The gel was dyed for 45 min in GelRed solution. GeneRuler DNA Ladder Mix (Thermo Scientific) was used to determinate the size of the bands. Representative PCR amplicons were excised from the gel and sequence verified by DNA sequencing (Seqlab Microsynth).