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A strand-specific polymerase chain reaction product is then generated to provide a suitable DNA template for quantitative methylation analysis using Ms-SNuPE.
Synthesized cDNAs were used as a template for quantitative real-time RT-PCR (qRT-PCR) with SYBR® Premix Ex Taq™ II (Takara, Shiga, Japan) and the Rotor-Gene 6000 (Corbett Research, Sydney, Australia).
The resultant cDNA was used as a template for quantitative PCR amplification in a Thermal Cycler Dice Real Time System II (Takara Biotechnology, Dalian, China) using SYBR Green I (Toyobo) as a fluorescent reporter.
Total RNA (200 ng) was used as the template for quantitative real-time RT-PCR analysis using the Brilliant SYBR Green QRT-PCR Master Mix (Stratagene, La Jolla, CA, USA), and PCR reactions were performed using a Multiplex 3000P Real-Time PCR System (Stratagene).
Resulting cDNA was used as a template for quantitative PCR (qPCR).
The RT products were used as template for quantitative real-time PCR using a Light-Cycler instrument (Roche).
One fiftieth of the cDNA was used as a template for quantitative real-time PCR (q-RTPCR).
1 100 dilutions of cDNA were used as template for quantitative PCR using iQ-SYBR Green Master Mix (Biorad) in a Biorad Opticon 2 cycler.
The cDNA reactions were then diluted to a volume of 50 µl with water and used as a template for quantitative PCR.
Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes.
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The resulting cDNA served as the template for Quantitative-PCR analysis using iTaq Universal SYBR Green Supermix (Bio-Rad) and ViiTM7 Real-Time PCR System Applied Biosystems Inc.., Foster City, CA).
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