Total RNA from OVCAR-3 and Caov-3 cells was extracted with Total RNA Isolating kit (BioTeke Corporation) following the manufacturer's protocols. Complementary DNA was obtained by RT using M-MLV reverse transcriptase kit (Takara Biotechnology Co., Ltd.) and RT Primer (GenScript) according to the manufacturer's protocol. The relative expression level of miR-638 was determined by qPCR amplification using TaKaRa Taq™ HS Perfect Mix (Takara Biotechnology Co., Ltd.) with SYBR Green (BioTeke Corporation). The thermocycling conditions consisted of: Pre-denaturation at 94˚C for 30 sec, followed by 40 cycles at 94˚C for 5 sec and 60˚C for 15 sec. miR-638 expression was normalized against the expression of U6. The relative expression level of miR-638 was converted to fold changes according to the 2-ΔΔCq method (24 (link)). The primers used were as follows: miR-638 forward, 5'-AATAGGGATCGCGGGCGG-3' and reverse, 5'-GCAGGGTCCGAGGTATTC-3', and U6 forward, 5'-GCTTCGGCAGCACATATACT-3' and reverse, 5'-GTGCAGGGTCCGAGGTATTC-3'.
Takara taq hs perfect mix
Manufactured by Takara Bio
Sourced in Japan
TaKaRa Taq™ HS Perfect Mix is a ready-to-use hot-start PCR mix that contains Taq DNA polymerase, buffer, dNTPs, and a proprietary anti-Taq antibody. It is designed to provide accurate and efficient DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase kit, by Takara Bio (1 mentions)
Sybr green, by BioTeke (1 mentions)
Rt primer, by GenScript (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Vazyme (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Rnase inhibitor, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
5 protocols using takara taq hs perfect mix
1
Quantification of miR-638 Expression
2
Genotyping of Chst14 Knockout Mice
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The method for PCR was reported in our previous study [18 (link)]. Pups were weaned and underwent ear punching (individual identification) at
3–4 weeks of age. The genotype of fetuses was analyzed on perinatal period (E18.5) with
tail samples. Genomic DNA was extracted from the tail and ear pieces of the mice using
MightyPrep reagent for DNA (Takara Bio Inc., Shiga, Japan). Primer sequences for wildtype
genotyping of exon 1 in the Chst14 gene were 5’-GGACCACCGCAGTGACTTG-3’
and 5’-ACAGGCATCCAATGCTCATTC-3’. Primer sequences for the neomycin resistance gene for
knockout genotyping were 5’-TGGCTCTCCTCAAGCGTATT-3’ and 5’-GTTTTCCCAGTCACGACGTT-3’. PCR
was carried out using TaKaRa Taq™ HS Perfect Mix (Takara Bio Inc.). The PCR conditions
were 94°C for 1 min and then 35 cycles of 94°C for 5 s and 65°C for 15 s. The PCR products
were analyzed by 1% agarose gel electrophoresis.
3–4 weeks of age. The genotype of fetuses was analyzed on perinatal period (E18.5) with
tail samples. Genomic DNA was extracted from the tail and ear pieces of the mice using
MightyPrep reagent for DNA (Takara Bio Inc., Shiga, Japan). Primer sequences for wildtype
genotyping of exon 1 in the Chst14 gene were 5’-GGACCACCGCAGTGACTTG-3’
and 5’-ACAGGCATCCAATGCTCATTC-3’. Primer sequences for the neomycin resistance gene for
knockout genotyping were 5’-TGGCTCTCCTCAAGCGTATT-3’ and 5’-GTTTTCCCAGTCACGACGTT-3’. PCR
was carried out using TaKaRa Taq™ HS Perfect Mix (Takara Bio Inc.). The PCR conditions
were 94°C for 1 min and then 35 cycles of 94°C for 5 s and 65°C for 15 s. The PCR products
were analyzed by 1% agarose gel electrophoresis.
3
MRSA Genomic DNA Extraction and mprF Gene Sequencing
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Genomic DNA was isolated from each MRSA strain as follows. Bacteria were cultured on trypticase soy agar at 37°C overnight. Bacterial colonies were then scraped from the agar plate, resuspended in 100 μL of lysis buffer (20 μg/mL lysostaphin, 1000 U/mL achromopeptidase in TE buffer), and incubated for 10 min at 37°C. Next, 50 μL of 0.5M KOH and 50 μL of 1M Tris/HCl were added for alkaline treatment and neutralization, respectively. The obtained solution contained DNA, which was used in subsequent experiments. Using 2 μL of the DNA aliquot as a template, PCR amplification of mprF was performed by TaKaRa Taq HS Perfect Mix (Takara Bio, Japan) with the primers listed in Table 2 . PCR clean-up was performed by ExoSAP-IT (Thermo Fisher Scientific, MA, USA), and the final product was used as a sequencing template. Sequence analysis was performed by Eurofins Genomics (Tokyo, Japan) with the primers listed in Table 2 . The sequencing data were analyzed using ApE software (created and maintained by Wayne Davis from the University of Utah) and aligned with the reference sequence of mprF of Staphylococcus aureus N315 (GenBank accession number NC_002745.2).
4
Reverse Transcription-PCR Analysis of Gene Expression
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Total mRNA was extracted from several cultured cells using the Total RNA Kit II (Omega, Norcross, GA, USA) according to the manufacturer's instructions. Then, 1 μg of total RNA was used to synthesize cDNA using the PrimeScript RT Reagent Kit (Vazyme Biotech Co., Nanjing, China). The cDNA of primary human RPE cells was generously gifted from Dr. Gu.22 (link) Semiquantitative reverse transcription PCR and real-time PCR was performed using TaKaRa Taq HS Perfect Mix (Takara Bio, Kusatsu, Japan) and ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech), respectively.
The primers used in this study were designed using the NCBI Primer BLAST system and the primers sequences are detailed in theTable .
PCR products were confirmed by agarose gel electrophoresis and melting temperature. The density analysis was performed by Image J (National Institutes of Health, Bethesda, MD, USA).
The primers used in this study were designed using the NCBI Primer BLAST system and the primers sequences are detailed in the
PCR products were confirmed by agarose gel electrophoresis and melting temperature. The density analysis was performed by Image J (National Institutes of Health, Bethesda, MD, USA).
5
Quantitative RT-PCR Analysis of Fracture Callus
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The fracture callus was collected and the total RNA was extracted by the RNApure high purity and rapid total RNA extraction kit (Bioteke Corporation, Beijing, China). The cDNA was synthesized by the M-MLV reverse transcriptase (Takara, Beijing, China) in the presence of RNase inhibitor (Takara) as per the user’s manual. The quantitative real-time PCR reaction system includes 200 ng cDNA, forward primer, and reverse primer 0.2 μg for each, 25 μl TaKaRa Taq™ HS Perfect Mix (Takara). The PCR was carried out on Exicycler 96 amplifier (Bioneer, Daejeon, Korea). The relative mRNA level was calculated using the 2-ΔΔCt method. The primers were as follows: HIF-1α (Forward: 5’-CTACTATGTCGCTTTCTTGG-3’, Reverse: 5’-GTTTCTGCTGCCTTGTATGG-3’); VEGF (Forward: 5’-CGGACAGACAGACAGACACC-3’, Reverse: 5’-AGCCCAGAAGTTGGACGAAA-3’); RUNX2 (Forward: 5’-CCATAACGGTCTTCACAAATC-3’, Reverse: 5’-GAGGCGGTCAGAGAACAAACT-3’); Osterix (Forward: 5’-AAAAGGAGGCACAAAGAAGC-3’, Reverse: 5’-GGGAAAGGGTGGGTAGTCAT-3’); COL1A1 (Forward: 5’-TCCTGCCGATGTCGCTATCC-3’, Reverse: 5’-TCGTGCAGCCATCCACAAGC-3’); SDF-1α (Forward: 5’- GCATCAGTGACGGTAAGC-3’, Reverse: 5’- GAAGGGCACAGTTTGGAG-3’); CXCR4 (Forward: 5’- GGCAATGGGTTGGTAATC-3’, Reverse: 5’- GACAATGGCAAGGTAGCG-3’), β-actin (Forward: 5’- GGAGATTACTGCCCTGGCTCCTAGC-3’, Reverse: 5’- GGCCGGACTCATCGTACTCCTGCTT-3’).
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