Extracta dna prep for pcr tissue

Fetal sex was confirmed by PCR amplification of the male-specific Sry gene (GenBank: X67204) from fetal legs. DNAs were extracted with Extracta DNA Prep for PCR-Tissue (Quanta BioSciences). PCR reactions were performed using AccuStart PCR SuperMix Kit (Quanta BioSciences) according to the protocol of the manufacturer with 0.04 nM of each Sry primer (forward 5′-TATGGTGTGGTCCCGTGGTG-3′; reverse 5′-ATGTGATGGCATGTGGGTTCC-3′), resulting in a 282-nucleotide amplicon. The following PCR conditions were used: 94 °C for 5 min and 72 °C for 10 min followed by 34 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min. Final extension was done at 72 °C for 10 min. Agarose gel electrophoresis was used for amplicon visualization.

Fetal sex was determined by PCR amplification of the male-specific Sry gene (GenBank no. X67204) from fetal legs. DNA was extracted with Extracta DNA Prep for PCR – Tissue (Quanta BioSciences) as described by the manufacturer. PCR amplification was performed using AccuStart PCR SuperMix Kit (Quanta BioSciences) with 0.04 nM of each Sry primer (forward: 5′TATGGTGTGGTC CCGTGGTG-3′; reverse: 5′-ATGTGATGGCATGTGGGTTCC-3′), resulting in an amplicon of 282 nucleotides. The following PCR conditions were used: 94°C for 5 min and 72°C for 10 min followed by 34 cycles of 94°C for 1 min, 65°C for 1 min and 72°C for 1 min. Final extension was done at 72°C for 10 min. Agarose gel electrophoresis was used for amplicon visualization. Sex of neonates was determined by examination of the ano-genital distance and gonadal morphology.

Genotyping was performed as described previously [49 (link)]. Genotyping samples were collected at weaning and/or during dissection. Ear or tail samples were routinely collected for DNA analysis. Extracta DNA Prep for PCR-tissue (Quanta Biosciences, Gaithersburg, MD, USA) was used to isolate genomic DNA from each mouse. Genotyping of each animal was performed using AccuStart II GelTrack PCR SuperMix (Quanta Biosciences, USA). After PCR, samples were analyzed on a 2% agarose gel by gel electrophoresis in tris-acetic acid-EDTA (TAE) buffer (Elatus Media Kitchen, University of Helsinki). Genotyping primers used are summarized in Table 1.