DNA extraction was performed according to the protocol of the Fast Tissue-to-PCR Kit, using mouse ears. Two μl DNA extracts were added to a 20 μl PCR reaction system containing Dream Taq PCR Master Mix primers and nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). DNA amplification was conducted in a Mastercycler personal PCR machine (Eppendorf, Germany), followed by separation on a 2% agarose gel containing DNA Stain G (SERVA, Heidelberg, Germany) under standard DNA electrophoresis conditions and UV illuminator visibility. The primers used in the PCR reaction are listed: Atg7 forward primer: 5′-TGGCTGCT-ACTTCTGCAATGATGT-3′, reverse primer: 5′-CAG-GACAGAGACCATCAGCTCCAC-3′; p48-cre forward primer: 5′-ACCGTCAGTACGTGAGATATCTT-3′, reverse primer: 5′-ACCTGAAGATGTTCGCGATTATCT-3′. We did not show genotyping from the Lamp2 mice.
Mastercycler personal pcr machine
Manufactured by Eppendorf
Sourced in Germany
The Mastercycler personal PCR machine is a compact and reliable thermal cycler designed for basic PCR applications. It features a high-quality, precise temperature control system to ensure accurate and reproducible results. The Mastercycler is a versatile tool suitable for a wide range of PCR-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dream taq pcr master mix primers, by Thermo Fisher Scientific (2 mentions)
Dna stain g, by Serva Electrophoresis (2 mentions)
First strand cdna synthesis kits, by Eppendorf (2 mentions)
Omniscript reverse transcriptase kit, by Qiagen (1 mentions)
Stratagen mx3000p, by Agilent Technologies (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Brilliant 2 sybr green qpcr master mix, by Agilent Technologies (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
6 protocols using mastercycler personal pcr machine
1
Mouse Ear DNA Extraction and Genotyping
2
Quantitative PCR Analysis of COX-1 and COX-2
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PBMC was lysed in RLT buffer (Qiagen, Hilden, Germany) supplemented with 1% 2-mercaptoethanol and stored in −80° until use. RNA was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instruction. The RNA quality and quantity was determined by spectrophotometry, based on the A260/A280 ratio (1.7–2.1 in all samples). Reverse transcription of RNA into cDNA was carried out using the Omniscript Reverse Transcriptase Kit (Qiagen) and Oligo(dT) primer (DNA Technology) in a Mastercycler personal PCR machine (Eppendorf AG, Hamburg, Germany). Real-time PCR was performed in a Stratagen Mx3000P (Agilent Technologies Inc., Santa Clara, CA) using Brilliant II SYBR® Green QPCR Master Mix (Agilent Technology) to detect COX-1 (PTGS1; Hs00924808_ml) and COX-2 (PTGS2; Hs00153133_ml). The thermal cycler was programmed as followed: initial set up for 10 min at 95°C, 45 cycles of denaturation at 95°C for 15 s each and thereafter annealing/extension at 60°C for 1 min. The gene expression was assessed using the comparative threshold cycle (Ct) method [12] (link).
3
Genotyping Mouse Samples via PCR
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DNA extraction was accomplished according to the protocol of the Fast Tissue-to-PCR Kit, using mouse tails. Four microliters DNA extracts were added to a 25 μl PCR reaction system containing Dream Taq PCR Master Mix primers and nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). DNA amplification was conducted in a Mastercycler personal PCR machine (Eppendorf, Germany), followed by separation on a 2% agarose gel containing DNA Stain G (SERVA, Heidelberg, Germany) under standard DNA electrophoresis conditions and UV illuminator visibility. The primers used in the PCR reaction are listed: Atg7 forward primer: 5′-TGGCTGCTACTTCTG-CAATGATGT-3′, reverse primer: 5′-CAGGACAGAGACCATCAGCTCCAC-3′ p48-cre forward primer: 5′-ACCGTCAGTACGTGAGATATCTT-3′, reverse primer: 5′-ACCTGAAGA-TGTTCGCGATTATCT-3′ Rip3 primer-1: 5′-CGCTTTAGAAGCCTTCAGGTTGAC-3′, Rip3 primer-2: 5′-GCCTGCCCATCAGCAACTC-3′, Rip3 primer-3: 5′-CCAGAGGCCACTTGT-GTAGCG-3′.
4
Quantitative Real-Time PCR Protocol for Gene Expression
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Total RNA was purified from fresh mouse liver tissue (50–100 mg) on ice with RNA fast 1000. The purity of the total RNA was checked with a spectrophotometer and the wavelength absorption ratio (260/280 nm) was between 1.7 and 2.0 in all preparations. Reverse transcription of total RNA (0.1–5 μg) to cDNA was performed using Revert Aid First Strand cDNA Synthesis Kits in 20 μL reaction volumes at 44 °C for 1 h using a Mastercycler personal PCR machine (Eppendorf AG, Hamburg, Germany). Specific primers were designed using the Beacon Designer 4.0 software and synthesized by Sunbiotech (Beijing Sunbiotech CO., Ltd, China). Real-time PCR was performed with Maxima SYBR Green/ROX qPCR Master Mix (2×) in an IQ 5.0 system. The system automatically monitors the binding of the fluorescent dye SYBR® Green to double-stranded DNA during each cycle of PCR amplification. PCR was performed under the following conditions: (1) heating at 50 °C for 2 min; (2) followed by 95 °C for 10 min and (3) 40 PCR cycles at 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCt) equation. The β-actin gene was used as a reference for normalizing the data. The derived normalized values are the mean of three runs. The mouse and human primer sequences are listed in Table 1 .
5
Validation of Microarray Findings by qPCR
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To confirm expression changes detected by microarray analysis, qPCR was performed on genes of interest (n = 10 per group). Target genes were Pdgfrb, Grm3, Flt1, Penk, and Nr1d1. Two stable reference genes were used to normalize relative expression results of genes of interest; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz), and peptidylprolyl isomerase A (Ppia). Primer information can be viewed in Supplementary Table 2 .
A SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) was used to reverse transcribe 100 ng of RNA to cDNA in an Eppendorf MasterCycler Personal PCR Machine (Eppendorf, Hamburg, Germany) with poly-T 20mer primers. The thermal profile used is as follows: an initial melting step of 95°C for 30 s, followed by 40 cycles of a 5-s 95°C melt, a 20-s 58°C annealing step, and a 20-s 72°C elongation step. A melt curve was performed from 60–95°C at 5-s 0.5°C increments to confirm specificity of primer binding, and relative expression values were calculated with REST 2009.
A SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) was used to reverse transcribe 100 ng of RNA to cDNA in an Eppendorf MasterCycler Personal PCR Machine (Eppendorf, Hamburg, Germany) with poly-T 20mer primers. The thermal profile used is as follows: an initial melting step of 95°C for 30 s, followed by 40 cycles of a 5-s 95°C melt, a 20-s 58°C annealing step, and a 20-s 72°C elongation step. A melt curve was performed from 60–95°C at 5-s 0.5°C increments to confirm specificity of primer binding, and relative expression values were calculated with REST 2009.
6
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was purified from fresh mouse liver tissue (50-100 mg) on ice with RNA fast 1000. The purity of the total RNA was checked with a spectrophotometer and the wavelength absorption ratio (260/280 nm) was between 1.7 and 2.0 in all preparations. Reverse transcription of total RNA (0.1-5 μg) to cDNA was performed using Revert Aid First Strand cDNA Synthesis Kits in 20 μL reaction volumes at 44 °C for 1 h using a Mastercycler personal PCR machine (Eppendorf AG, Hamburg, Germany). Specific primers were designed using the Beacon Designer 4.0 software and synthesized by Sunbiotech (Beijing Sunbiotech CO., Ltd, China). Real-time PCR was performed with Maxima SYBR Green/ROX qPCR Master Mix (2×) in an IQ 5.0 system. The system automatically monitors the binding of the fluorescent dye SYBR® Green to double-stranded DNA during each cycle of PCR amplification. PCR was performed under the following conditions: (1) heating at 50 °C for 2 min; (2) followed by 95 °C for 10 min and (3) 40 PCR cycles at 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCt) equation. The β-actin gene was used as a reference for normalizing the data. The derived normalized values are the mean of three runs. The mouse and human primer sequences are listed in Table 2 .
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