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An amount of 200 ng of sample was fragmented on the Covaris S2 in microtubules.
The reaction system included 150 ng of sample total RNA and 1 μl of the master mix plus RNase-free water to bring the final volume to 5 μl.
Approximately 50 ng of sample cDNA per well were used for the analysis, except for the case of cDNA from the SC population from a control individual, for which the amount of cDNA had to be reduced to 7.5 ng per wall to prevent overloading of the assay.
Approximately 30 ng of sample DNA per well were used in the analysis, except for the case of DNA from the additional clones from individual CES6 for which only 5 ng per well were used due to the low amount of available DNA.
For the 2-step PCR protocol, the first PCR was done in a volume of 20 μl containing 10 ng of sample DNA, 10 μl of 2 × Phusion High-Fidelity PCR Master Mix and 0.375 μM of each locus-specific primer, supplemented with water to the final volume.
The 30 µl reaction mixture contained 1 μl (30 ng) of sample DNA, 1x PCR buffer, 1 mg/ml of BSA, 0.2 mM dNTP's, 0.3 μM of each primer and 1250 U/ml GoTaq DNA Polymerase (Promega, WI, USA) in a 30 μl reaction volume.
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100 ng of samples with 260/280 readings of >1.8 and 260/230 readings of >1.9 were subsequently converted into cDNA using RT2 First strand kit according to the manufacturer's instruction.
Briefly, 750 ng of test sample cRNA was mixed with 750 ng of reference sample cRNA, in the presence of target controls.
Briefly, 750 ng of test sample cRNA was mixed with 750 ng of reference sample cRNA in the presence of target controls.
The samples were then immediately hybridized onto HT-12_v3_BeadChips; 750 ng of each sample was loaded onto the arrays.
Each of the 96 samples in each plate were quantified using Qubit® and 25 ng of each sample used to compile a library.
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