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This included a 30 seconds denaturation step at 95°C, a 30 seconds annealing step at 55°C (Figα) or 57°C (PAR6 and β-actin), and a 40 seconds extension step at 72°C.
The initial PCR activation step was at 94°C for four minutes, followed by the denaturation step at 94°C for one minute, primer-annealing step at 55°C for 30 seconds, extension step at 72°C for one minute, and the final extension step at 72°C for ten minutes.
PCR was performed at 55 degrees Celsius, 32 amplification cycles, 60 seconds extension step.
The Tc_CLB1 amplicon of 3 kb size uses a 45 seconds extension step, in comparison with the other amplicons that uses just 10 seconds.
After 6-minute initial denaturation at 96°C, a "hot start" amplification was initiated by adding DreamTaq DNA polymerase, followed by 19 cycles consisting of a 15 seconds denaturation step at 95°C, a 30 seconds annealing step at 64°C, and a 15 seconds extension step at 72°C, with a final extension for 45 minutes at 72°C.
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Samples were run using the Lightcycler programme, with a 15-minute incubation at 95°C followed by 45 thermal cycles, consisting of a 15-second denaturation step at 94°C, then a 20-second annealing step at 60°C, and a 20-second extension step at 72°C after which fluorescence was read.
8) The second primer-extension reaction using the fluorescently labelled 3′- O- 2-cyanoethyl -dNTP derivatives 2– 5 and the reverse transcriptase was carried O- 2-cyanoethyl -dNTPn cO- 2-cyanoethyl -dNTPO- 2-cyanoethyl -dNTPn step (step 2).
PCR was performed for 40 cycles comprising a 60-second denaturation step at 94°C, a 60-second annealing step at 55°C, a 90-second extension step at 72°C followed by 10 minutes at 72°C.
Quantitative PCR was performed with the following programme: pre-incubation at 50°C for 2 minutes, initial denaturation at 95°C for 5 minutes, 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, elongation at 72°C for 30 seconds, and a 20-second final extension step.
Real-time PCR reactions were performed at 95°C for 15 minutes, followed by 40 cycles of amplification consisting of denaturation step at 95°C for 15 seconds and extension step at 60°C for 1 minute.
For fluorescence signal acquisition, time and temperature profile were set as follow: holding step at 95°C for 15 minutes for enzyme activation, 40 cycles starting in denaturation step at 95°C for 10 seconds, annealing at 58°C for 10 seconds and lastly extension step at 72°C for another 10 seconds.
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