DNA was extracted and purified as previously described by Yehia et al. [8 (link)] with minor modifications. Five to ten cryosections of 10 μm were harvested from each OCT block in precooled tubes and lysed in 400 μl cell lysis solution (Qiagen). 30 μg of proteinase K was added to each sample. Following brief vortexing, the samples were incubated overnight under shaking at 56°C. After a 30-min incubation at 37°C with RNAse A (20 μg/tube), samples were spun at 13,000 g for 5 min to remove residual debris. The supernatant was collected and cooled on ice for 1 min, and 133 μl of protein precipitation solution (Qiagen) were added. After vortexing, samples were left on ice for at least 20 min and then centrifuged at 13,000 g for 5 min. The supernatant containing DNA was transferred to a new tube, and the DNA was precipitated by the addition of 400 μl of 100% isopropanol and 1 μl of glycogen solution (20 mg/ml; Qiagen). After mixing and centrifugation at 13,000 g for 5 min, the pellet of DNA was washed twice with 70% ethanol, dried and resuspended in 30 μl of 10 mM Tris buffer, pH 8.5.
Glycogen solution
Manufactured by Qiagen
Glycogen solution is a lab equipment product used as a carrier to facilitate the precipitation and recovery of nucleic acids, such as DNA and RNA, from samples. It is a highly purified glycogen preparation that can be used to improve the efficiency and yield of nucleic acid isolation and purification procedures.
Automatically generated - may contain errors
3 protocols using glycogen solution
1
DNA Extraction from Cryosections
2
Extracting High-Quality gDNA from Frozen Hematoma
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Frozen human fracture hematoma samples (n = 8) were thawed in a 37°C water bath and further kept on ice. To dissolve the hematoma samples, Clotspin® Baskets from Qiagen were used according the manufacturing protocol: Purification of archive-quality gDNA from clotted whole blood using Clotspin® Baskets and the Gentra® Puregene® blood kit provided by Qiagen. All protocol steps were followed according to the manufacturer's instructions. Steps included the usage of isopropanol and Glycogen Solution (20 mg/ml from Qiagen) and washing of gDNA pellets with 70% ethanol. gDNA was further air dried at room temperature until no remaining fluids remained. gDNA was incubated with 500 μl DNA hydration solution provided within the kit (www.qiagen.com/literature/handbooks/default .aspx). gDNA was incubated at 65°C until it was dissolved.
gDNA quality and quantity were confirmed using the NanoDrop-ND-1000 system (PEQLAB GmbH). Samples were stored at −80°C until further experiments.
gDNA quality and quantity were confirmed using the NanoDrop-ND-1000 system (PEQLAB GmbH). Samples were stored at −80°C until further experiments.
3
Genomic DNA Extraction from Sperm
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Genomic DNA from sperm samples was extracted using the Puregene Core Kit A (QIAGEN, #1042601) according to the manufacturer's instructions with minor modifications previously described in Arbeithuber et al. (2015) (link). In brief, DNA was extracted from a saliva swab or 25 µl of sperm (∼106 sperm cells) with the addition of 0.5 µl of Proteinase K solution (20 mg/ml) and 6 µl of 1 M DTT, followed by an overnight incubation step at 37 °C. During DNA precipitation, 0.25 µl of glycogen solution (QIAGEN, #1045724) were added. Vortexing steps were replaced by intensive manual shaking for 1 min.
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