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The clones were validated by restriction digestion/sequencing and then sub-cloned in a pcDNA3.1 vector (Invitrogen) to generate expression constructs.

All clones were validated by sequencing.

The resulting expression clones were validated by restriction digest analysis and DNA sequencing.

Positive isolated clones were validated by PCR, qPCR and resequencing of the long amplicon.

Clones were validated by PCR with primers specific for pJK148 (Supplementary Figure S10).

Positive clones were validated by PCR and restriction enzymes digestion and sequenced by Shanghai Ying-Jun BioTech.

The TCR specificity of clones was validated by FACS using staining with mAbs specific for CD3, Vα24, Vβ11 and the invariant iNKT cell TCR chain (6B11), and the cell surface phenotype was determined by staining with CD4 and CD8α specific mAbs.

miR-205 expression in H358 and H441 shCtrl and shTMP4 cell clones was validated by qPCR (Supplementary Figure 1B).

The anchoring of the markers on the positive BAC clones was validated by direct PCR with 1 μl of bacterial solution.

The infectivity of the BAC DNA clone was validated by MDV reconstitution from chicken embryo fibroblasts transfected with MDV-BAC DNA, which was extracted from electroporated Escherichia coli DH10B cells.

This clone was validated as a Y-linked BAC by a male-specific PCR (Fig. 1).