After trypsinization, the ES cell pellets were lysed with 0.1 mg/ml proteinase K in lysis buffer (0.1 M Tris pH 8.5, 5 mM ethylenediaminetetraacetic acid, 0.2% sodium dodecyl sulfate [SDS], 0.2 M NaCl), and the DNA was isolated through ethanol precipitation. E6.5–E8.5 DNA was isolated using 40 μl of QuickExtract (Lucigen, WI) solution according to the manufacturer's instructions, and 1–5 μL of the lysate was used for PCR.
PCR was used to amplify a region in Nhlrc2, where the insertion of the targeting construct resulted in a new SacI digestion site, with the primers shown in Table2 . Phire Hot Start II Polymerase (Thermo Fisher Scientific, Waltham, MA) and a Piko Thermal Cycler (Thermo Fisher Scientific, Vantaa, Finland) were used according to the manufacturer's instructions. Sanger sequencing was performed to validate the PCR product as described previously (Hiltunen et al., 2020 (link)). The PCR product from E6.5–8.5 embryos was precipitated with NaCl (4 M, 1:10) and ethanol before digestion overnight at −20°C or for 30 min at −70°C. The PCR product was digested using the SacI restriction enzyme (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer's instructions and run on 1.5% agarose gel (BioNordika, Helsinki, Finland). SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA) was used for detection.
PCR was used to amplify a region in Nhlrc2, where the insertion of the targeting construct resulted in a new SacI digestion site, with the primers shown in Table
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