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Band sizes ranging from 80-100 bp were cut from the gel and purified.
Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 μl EB.
cDNA fragments with 200 ± 25 bp were cut from the gel and purified using Qiagen gel purification kit (Cat. No. 28704, Qiagen, Valencia, CA, USA).
Bands of the expected size (671 bp) were cut from agarose gels and purified and cloned as described above; four clones from each genotype were sequenced.
Fragments with insert sizes of 200 to 400 bp were cut from the gel, and DNA was extracted using QIAquick Gel extraction kit (ref. 50928706, Qiagen) and eluted in 20 μl EB.
The fragments of 350 400 bp were cut from the gel, purified (gel extraction kit, Qiagen GmbH, Hilden, Germany) and sent for sequencing (Roche/454 sequencing, dr. Elio GWM Schijlen, Plant Research International, Wageningen, The Netherlands).
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The resulting sample was run on a 6% native PAGE gel, and the 150-175-bp size range (corresponding to 60-85 bp) was cut from the gel, shredded, and extracted overnight in WT-Seq PAGE elution buffer.
The linked DNA was purified with an Agencourt AMPure XP kit (Beckman Coulter) before running the DNA on a 3% low-range agarose gel (Bio-Rad) and the region at ~125 200 bp was cut from the gel under blue light and re-purified using a Wizard® SV Gel extraction and PCR clean up kit (Promega).
After fractionation on a 1% agarose gel, the DNA bands corresponding to 236 bps were cut from the gel, isolated using GenElute Spin columns (Supelco), and ethanol-precipitated.
The bands of 1,400 bp were cut out from the agarose gel, and the gel slices were purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA).
The last 5 bp were cut off from unmapped tags for the next matching with chromosome and exon junction database (allowing two mismatches) to increase annotation rate of raw data.
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