Polymerase chain reaction (PCR) was performed using Taq polymerase-containing master mix (FastStart PCR Master; Roche, Basel, Switzerland), with 20nM forward and reverse primers (5′-TCCAGAATCTGTTCCAGAGCGTGC -3′ and 5′-GCTGTGAAGGTTGCTGTTCCTCAT -3′, respectively). PCR products were analyzed on a 20% Tris-borate-EDTA gel (Invitrogen). The Big Dye cycling protocol (Invitrogen) was performed using a two step method without the annealing step (96°C 10 s, 60°C 4 min).
Tris borate edta gel
Manufactured by Thermo Fisher Scientific
Tris-borate-EDTA (TBE) gel is a type of electrophoresis gel used for the separation and analysis of nucleic acids, such as DNA and RNA. It is a buffer solution that provides the necessary ionic environment for the migration of charged molecules through the gel matrix under the influence of an electric field.
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2 protocols using tris borate edta gel
1
Polymerase Chain Reaction Protocol
2
NF-κB Nuclear Translocation Assay
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Nuclear extracts of the HK-2 cells were prepared with the NE-PER nuclear extraction reagent (Pierce Biotechnology). The biotin-labeled NF-κB oligonucleotide sequence was 5′-biotin-AGTTGAGGGGACTTTCCCAGGC-3′. The binding reactions contained 10 µg of the nuclear extract protein, buffer (10-mM Tris [pH 7.5], 50-mM KCl, 5-mM MgCl2, 1-mM dithiothreitol, 0.05% Nonidet P-40, and 2.5% glycerol), 1 µg of poly (dI-dC), and 2-nM biotin-labeled DNA. The reactions were incubated at 23 °C for 20 minutes. The competition reactions were performed by adding 10-fold excess unlabeled double-stranded NF-κB consensus oligonucleotides to the reaction mixture. The reactions were electrophoresed on a 6% precasted Tris-borate–EDTA gel (Invitrogen) at 100 V for 1 hour 30 minutes in a 100-mM Tris-borate–EDTA buffer. The reactions were then transferred to a nylon membrane. The biotin-labeled DNA was detected with a LightShift chemiluminescent electrophoretic mobility shift assay kit (Pierce Biotechnology).
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