All manipulations should be performed in a clean room or a PCR hood. Use only certified DNA-free tubes and reagents and wash surfaces likely to come into contact with the samples in 0.05% hypochlorite bleach. Rinse thoroughly with DNA-free water.
1. Remove one or two 1.2 mm discs from the card-stored sample and place into a thin-walled PCR tube or a 96-well tray.
For the best results, punch in the center of the area where the sample was applied.
2. Wash the disk in 100 µl of DNA-free water by incubating at room temperature for 15 minutes. Aspirate the water from the disc and discard.
- 5 µl of 10x buffer MAGENTA
- 44 µl of DNA-free water
- 1 µl prepGEM™
2. Incubate at 75°C for 5 minutes.
3. Incubate at 95°C for 5 minutes.
A thermal cycler can be used for this step
4. Centrifuge for 5 minutes at 16,000 x g and transfer the supernatant to a fresh tube.
The DNA is in this solution - not the punch.
The sample is now ready for quantification and analysis.
Typically, 2 - 5 µl should be used in PCR
- Despite the distribution of the blood on the card appearing even, the density of cellular material is not. Some yield variation is to be expected.
- The Storage Card procedure can be automated using most liquid handling robots.
- The ZyGEM Storage Card kits are a preparative method for DNA extraction from most types of storage cards. Its purpose is to lyse cells and to strip the DNA of nucleoproteins. Extracted DNA can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications. All buffers and reagents are compatible and so downstream purification steps are not necessary
- Storage cards for cells and DNA contain preservatives that can seriously inhibit Taq DNA polymerase. The pre-soak step is to remove these inhibitors and is essential for reliable results.
- For accurate yield assessment, a qPCR is recommended. The DNA produced by the kits is approximately 90% single-stranded. If standard fluorescent chelating dyes are to be used for quantification, this factor should be taken into consideration.
- As with any preparative method for nucleic acid extraction, for best results prepare and manage samples at 4ºC, or on ice, before and after extraction.
- When storing the sample after extraction, aspirate the supernatant from the card punch.
- prepGEM / forensicGEM is stable at 4º C. However, once the tube has been opened and for longer-term storage we recommend storage at -20ºC.
- For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.