Sentence examples for three times subsequently from inspiring English sources

For expression of full-length human neurofibromin with C-terminal eGFP tag (synNF1eGFP) the eGFP coding sequence was cloned three times subsequently in frame at the 3′-end of the full-length NF1 sequence in the synNF1 pFastBacHta vector.

The samples have been rinsed three times subsequently with pure toluene for 5 min in each case and then dried with compressed air. Figure 1 Schematic view of the biofunctionalization of ZnO.

The Corvis ST measurements were repeated on the patients three times subsequently on the same patient by the same operator (Roberta Calienno) Two surgeons performed all surgical procedures (Leonardo Mastropasqua and Mario Nubile).

These ear samples were homogenized in 500  μl of ice-old PBS, snap frozen and thawed four times, subsequently submitted to sonication (twice for 10 s) and centrifuged at 10 000 × g for 15 min at 4 °C.

Liposome-containing solutions were frozen and thawed three times, and subsequently passed through an extruder (Avanti Polar Lipids) with a 400 nm membrane.

The membranes were washed three times and subsequently incubated at room temperature for 1 h with anti-mouse or anti-rabbit immunoglobulins-horseradish peroxidase in 5% (w/v) non-fat milk in PBS-T.

Serum samples were diluted to 1 100 with distilled water, incubated with antigen for 30 min at room temperature, washed three times, and subsequently incubated for another 30 min with antihuman IgA.

The PVDF membranes were washed by TBST for three times and subsequently incubated with second antibody HRP (horseradish peroxidase) goat anti-rabbit IgG (immunoglobin G) or HRP goat anti-mouse IgG, respectively (Zhongshan Goldenbridge Biotechnology).

After lysing, slides were washed with enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, pH 8; 0.2 mg/mL bovine serum albumin, BSA) for three times and subsequently incubated with 30  μL of fpg enzyme (1 : 3000 dilution in enzyme buffer) for 30 min at 37°C.

Wells were washed with 0.05% (v/v) Tween in PBS four times, and subsequently blocked with 200 μL 0.05% (v/v) Tween and 1% (v/v) Bovine Serum Albumin (BSA) in PBS for 1 h at 37 °C.

Afterwards, cells were washed three times with PBS and subsequently fixed with 2.5% glutaraldehyde in 0.03 M potassium phosphate buffer for at least 24 h.