Extraction of DNA from Frozen Islet Repository Samples
IIDP-HIPP
Abstract
This Standard Operating Procedure (SOP) provides Human Islet Phenotyping Program (HIPP) procedures for DNA extraction from frozen islet pellets using the Promega Wizard® Genomic DNA Purfication Kit.
This SOP defines the assay method used by the Human Islet Phenotyping Program (HIPP) for quantitative and qualitative determination of the Purified Human Pancreatic Islet product, post-shipment, manufactured for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP).
Steps
Procedures
Assemble all items described in Materials .
Make Nuclei Lysis Buffer: Mix 500µL Nuclei Lysis Solution with 120µL 0.5M EDTA PH 8.0. Chill on ice.
Add 600µL chilled Nuclei Lysis Buffer and 20µL proteinase K to the islet pellet. Vortex vigorously for 10 seconds. Incubate at 65°C for 30 minutes.
Add 3µL RNase Solution to the islet lysate and mix by inverting the tube 2-5 times. Incubate at 37°C for 30 minutes. Cool to room temperature.
Add 200µL Protein Precipitation Solution. Vortex and incubate on ice for 5 minutes.
Centrifuge at 13,000-16,000 xg (maximum speed of microcentrifuge) for 5 minutes at 4°C .
Transfer supernatant to a clean 1.5 mL microcentrifuge tube containing 600µL room temperature isopropanol. Mix gently by inversion. Incubate at -20°C for 15 minutes.
Centrifuge at 13,000-16,000 xg for 1 minute at room temperature.
Remove supernatant and add 600µL room temperature 70% ethanol. Mix gently by inversion.
Repeat step 8.
Aspirate the ethanol and air-dry the pellet for 15 minutes.
Rehydrate the DNA in 100µL of DNA Rehydration Solution for 30 minutes at room temperature.
Use NanoDrop to measure DNA concentration.
Store the DNA at -20°C .
Deviations and Resolutions
Document any deviations that occurred during this protocol that affect the final results and report with the analysis of the assay.
Data Storage and Reporting
To facilitate data management and ensure data security, the Vanderbilt HIPP uses an institutional server-based platform for data storage and analysis.
- Upon completion of image acquisition and analysis, annotated images containing metadata, image analysis outputs, and documentation of any deviations and repetitions that are warranted will be uploaded to the IIDP-HIPP database and disseminated to IIDP-affiliated investigators and islet isolation centers.
