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The expression obtained is inserted into Eq. (27) and one obtains one equation with a 1 as the unknown.
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The resulting plasmid was digested with BamHI and EcoRI, and the fragment obtained was inserted into the BamHI-EcoRI sites of the pUG36 yeast expression vector.
cDNAs of hARD1 and long MLCK variant 1/2 (NM_053026, NM_053026) were cloned by reverse transcription and PCR using Pfu DNA polymerase, and the cDNAs obtained were inserted into pcDNA vector by blunt-end ligation.
The products obtained were inserted into pGEM-T (Promega) and sequenced using the BigDye terminator v3 protocol (Applied Biosystems).
None of the sequences we obtained were inserted inside genes, and most were inserted into intergenic regions, sometimes very close to coding sequences.
The two xylanase genes obtained were inserted into pUCm-T and then transformed into E. coli JM109, respectively, followed by DNA sequencing.
Positive clones were digested with XhoI and PstI, and the fragments obtained were inserted in the corresponding sites of pEGFP-N1 (Clontech).
The resulting plasmid was digested with SmaI and SalI, and the obtained fragment was inserted into the SmaI- SalI sites of the pUG36 yeast expression vector to obtain the pYH2 construct.
The resulting plasmid was digested with SpeI and BamHI, and the obtained fragment was inserted into the SpeI- BamHI sites of the pUG36 yeast expression vector (Guldener and Hegemann, unpublished data) to obtain the pYH1 construct.
This quality control sample was analysed with each batch of samples and the obtained results were inserted into QC charts.
Full length amphiER were amplified by polymerase chain reaction (PCR) and the obtained fragments were inserted into a pSG5 vector between EcoR1 sites.
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