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An experiment was designed where skim milk was heated at high temperatures (76°C for 7 min) to stop the gravity separation of SC and then colostrum was added back to try to restore the gravity separation of SC in increments to achieve 0, 0.8, 0.8, 2.0, and 4.0 g/L of added immunoglobulins.
The mixtures were heated to 100 °C for 10 min to stop the reaction.
And then the mixtures were heated at 100 C for 5 min to stop the reaction.
After 2 h of incubation, the samples were heated at 95 °C for 5 min to stop the hydrolysis.
Moreover, the total duration of an attack is about 20 min, so the detection delay must be small enough (less than 1 min) to stop the attack quickly.
The samples were heated at 95 °C for 5 min to stop the reaction and were prepared for high-performance liquid chromatography (HPLC) analysis.
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The reaction times were: Figure 3A,B, Figure 3 figure supplement 2, and Figure 3 figure supplement 3: 90 min; Figure 3C: 60 min; Figure 3D: 120 min. To stop the reactions and to remove background signals the gels were subsequently extensively washed with a solution containing 5% (vol/vol) trichloroacetic acid and 1% (vol/vol) phosphoric acid for at least six times for 15 min each.
The constantly stirred reaction mixture was then heated to 95°C for 15 min. To stop the reaction, 280 mL of deionized water and 5 mL of H2O2 were added.
Reactions were performed at 30°C for 80 min. To stop the reaction, acetic acid was added to a final concentration of 10% (v/v).
All RNA samples were additionally treated with 2 U of DNase I (Promega) at 37°C for 30 min. To stop DNase I activity, DNase I inhibitor (Promega) was added to the reaction tube.
After washing the cells twice in 10 ml PBS, pH 7.4, they were incubated with 0.5 mM CFSE in 1 ml PBS per 107 cells at RT for 8 min. To stop the staining reaction 8 ml RPMI plus 10% FCS was added.
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