We determined the levels of the m.5024C>T mutation by “last-cycle hot” PCR25 (link) which visualizes only nascent amplicons and removes interference from hetero-duplexes formed during melting and annealing cycles. Total genomic DNA was extracted from FACS sorted cells with the NucleoSpin Tissue XS kit (740901.50; Takara) according to the manufacturer’s instructions and from tissues with phenol-chloroform extraction24 (link). The DNA concentration was determined spectrophotometrically (BioTek Synergy H1 hybrid). The WT allele for the m.5024C>T mutation completes a mismatched primer, creating a restriction site for PstI not present in the mutant PCR product. The following primers were used: F- 5′-CCACCCTAGCTATCATAAGCACA-3′ and B-5′-AAGCAATTGATTTGCATTCAATAGATGTAGGATGAAGTCCTGCA-3′. PCR products were digested with PstI and resolved in a 12% polyacrylamide gel. The radioactive signal was quantified using a Cyclone phosphor-imaging system (PerkinElmer) and OptiQuant software Version 5.0 (PerkinElmer).
Cyclone phosphor imaging system
Manufactured by PerkinElmer
The Cyclone phosphor-imaging system is a lab equipment product that captures and analyzes data from radiolabeled samples. It uses phosphor imaging technology to detect and quantify the radioactive signals from these samples.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin tissue xs kit, by Takara Bio (2 mentions)
Optiquant software version 5, by PerkinElmer (2 mentions)
Synergy h1 hybrid, by Agilent Technologies (2 mentions)
Dreamtaq green buffer, by Thermo Fisher Scientific (1 mentions)
Neg013h100uc, by PerkinElmer (1 mentions)
Dreamtaq polymerase, by Thermo Fisher Scientific (1 mentions)
5 protocols using cyclone phosphor imaging system
1
Quantifying Mitochondrial Mutation Levels
2
Brd4 PDID Phosphorylation Assay
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Purified bacterial Brd4 phosphorylation-dependent interaction domain (PDID) proteins were used for this study (PDID wild-type, PDID S484/488 A, PDID S492/494 A). In each reaction, 100 ng of PDID protein was incubated with 200 μM of ATP, 1000U of CSNK1D (NEB, P6030S), and 1X NEBbuffer for protein kinases for 30 min at 30 °C. Laemmli sample buffer was added to terminate the reactions, and the samples were heated to 95°C and resolved by SDS-PAGE. The resolved bands were transferred onto a nitrocellulose membrane and subjected to western blotting with the appropriate antibodies.
In the case of the radioactivity assay, 5 μCi [γ-32P]ATP (Perkin–Elmer, BLU002H250UC) was added to each reaction. The resolved SDS-PAGE gel was exposed, and radioactive signal was quantified using a Cyclone phosphor imaging system (Perkin–Elmer).
In the case of the radioactivity assay, 5 μCi [γ-32P]ATP (Perkin–Elmer, BLU002H250UC) was added to each reaction. The resolved SDS-PAGE gel was exposed, and radioactive signal was quantified using a Cyclone phosphor imaging system (Perkin–Elmer).
3
Quantifying Mitochondrial DNA Mutations
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Total DNA was extracted from flash-frozen tissues using phenol–chloroform and from FACS-sorted cells using the NucleoSpin Tissue XS kit (740901.50, Takara). DNA concentration was determined spectrophotometrically (BioTek Synergy H1 hybrid). Levels of the m.5024C>T mutation were determined by “last-cycle hot” PCR52 (link), wherein the last cycle of the PCR is run using radioactively labeled nucleotides. This method removes interference from heteroduplexes formed by previous melting and annealing steps by only allowing visualization of nascent amplicons. PCR amplicons were obtained with the following primers: F-5′-CCACCCTAGCTATCATAAGCACA-3′ and B-5′-AAGCAATTGATTTGCATTCAATAGATGTAGGATGAAGTCCTGCA-3′20 (link). RFLP analysis was done by digesting amplicons with PstI-HF (R3140S, New England BioLabs), which digests the WT mtDNA but not the mutant mtDNA carrying the m.5024C>T point mutation. After digestion, products were run in a 12% polyacrylamide gel and signal was detected using the Cyclone phosphor-imaging system (Perkin Elmer) and OptiQuant software Version 5.0 (Perkin Elmer)20 (link).
4
Viral Expression of Avpr1b in Mouse Hippocampus
5
Quantitative Analysis of Mitochondrial Mutations
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PCR amplicons were obtained using the following set of primers:
Forward: 5′-CCTAGCTATCATAAGCACA-3′
Backward: 5′-AAGCAATTGATTTGCATTCAATAGATGTAGGATGAAGTCCTGCA-3′
Amplicons were generated in a reaction containing 1× DreamTaq green buffer (Thermo Fisher Scientific), 75 nmol of each primer, 5 μmol of dNTPs, and 1 U DreamTaq polymerase (Thermo Fisher Scientific) in a final volume of 20 μL. Cycling conditions were as follows: 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 30 s, 59°C for 30 s, 72°C for 45 s, one cycle of 72°C for 4 min, and 12°C hold. Resulting PCR products (174 bp) were subjected to one additional cycle with dCTP, [α-32P] (Perkin Elmer-cat #NEG013H100UC),64 (link) which visualizes only nascent amplicons and removes interference from hetero-duplexes formed during melting and annealing cycles. Upon PCR amplification, the WT allele for the m.5024C>T mutation, together with a 1-bp mismatched oligonucleotide primer, creates a restriction site for PstI not present in the PCR product originated from the mutant mtDNA. Labeled products were digested with 0.5 μL PstI (NEB, 20U/μL). PstI generates 2 fragments (136 and 40 bp) and resolved in a 12% polyacrylamide gel. The radioactive signal was quantified using a Cyclone phosphor-imaging system (PerkinElmer) and OptiQuant software Version 5.0.14 (link)
Forward: 5′-CCTAGCTATCATAAGCACA-3′
Backward: 5′-AAGCAATTGATTTGCATTCAATAGATGTAGGATGAAGTCCTGCA-3′
Amplicons were generated in a reaction containing 1× DreamTaq green buffer (Thermo Fisher Scientific), 75 nmol of each primer, 5 μmol of dNTPs, and 1 U DreamTaq polymerase (Thermo Fisher Scientific) in a final volume of 20 μL. Cycling conditions were as follows: 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 30 s, 59°C for 30 s, 72°C for 45 s, one cycle of 72°C for 4 min, and 12°C hold. Resulting PCR products (174 bp) were subjected to one additional cycle with dCTP, [α-32P] (Perkin Elmer-cat #NEG013H100UC),64 (link) which visualizes only nascent amplicons and removes interference from hetero-duplexes formed during melting and annealing cycles. Upon PCR amplification, the WT allele for the m.5024C>T mutation, together with a 1-bp mismatched oligonucleotide primer, creates a restriction site for PstI not present in the PCR product originated from the mutant mtDNA. Labeled products were digested with 0.5 μL PstI (NEB, 20U/μL). PstI generates 2 fragments (136 and 40 bp) and resolved in a 12% polyacrylamide gel. The radioactive signal was quantified using a Cyclone phosphor-imaging system (PerkinElmer) and OptiQuant software Version 5.0.14 (link)
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