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The insert of both constructs were removed from the Vector backbone sequences by Not I digestion and gel purified prior to pronucleus injection.
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As in phase I, constructs were removed from confinement at t = 10 days, and cultured in individual wells for the remainder of the study.
Polymerization was initiated at 37°C and allowed to proceed over 30 min. After polymerization, the cell constructs were removed from the syringe and placed in 24-well plates containing specific medium (see below for details), for up to 10 days.
Polymerization was initiated at 37°C and allowed to proceed over 30 min. After polymerization, the cell constructs were removed from the syringe and placed in 24-well plates, containing specific medium, for up to 96 h.
Based upon prior results in our laboratory demonstrating enhanced tissue mechanical properties [4], self-assembled constructs were removed from confinement in the agarose well at 2 wks and transferred into 10-mm diameter wells coated with 2% agarose, where they remained up to 8 wks.
The adenoviral constructs were removed after transduction, and the cells were passaged in KGM-Gold.
After 6 h, the siRNA constructs were removed and cells were further incubated in SFM overnight.
After 1 and 2 weeks, the constructs were removed for histological analysis and evaluation of vascularization.
After 7 and 14 days after implantation, the constructs were removed.
For the separate isolation of RNA from the three classified groups of cells, the BNC-cartilage constructs were removed from the wells and the BNC insert was carefully removed with forceps.
Among 657 genes selected as candidates that are conserved in the A. thaliana genome, a total of 557 bait clones were used for screening (98 self-active clones and two failed constructs were removed).
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