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DNA Extraction Using
forensicGEM™ Storage Card Saliva
forensicGEM™ Storage Card Saliva is a kit specifically developed for extracting DNA from saliva stored on FTA cards. FTA cards are commonly used in forensic laboratories for storing reference samples – samples of saliva taken from buccal swabs.
- forensicGEM™ is validated and quality tested for forensic applications.
- The simplicity of the method maximizes yields of DNA from trace samples.
- The kit’s optimized methods and formulations provide DNA in just over 30 minutes.
- All buffers and reagents are PCR compatible with common profiling kits.
- The methods are closed-tube and hands-off.
These features make forensicGEM™ Storage Card Saliva particularly valuable for high-throughput automation of high-volume crime samples. The procedure is easily adaptable for 96-well plates and automation, and the closed-tube nature of the method protects the integrity of the sample through the extraction procedure.
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Workflow
Extraction Method
Sample Preparation
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.
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.
Uneven application of the swab onto the storage card results in DNA yield variations. For the best results, punch in the center of the area where the sample was applied.
Extraction Method
1. Add:
- 5 µl of 10x buffer SKY BLUE
- 44 µl of DNA-free water
- 1 µl forensicGEM™
2. Incubate at 75°C for 15 minutes.
3. Incubate at 95°C for 5 minutes. A thermal cycler can be used for this step
4. Pipette the solution to a new tube.
The DNA is in this solution - not the punch.
Technical Tips
- The forensicGEM™ Storage Card procedure can be automated using most liquid handling robots.
- forensicGEM™ Storage Card is a preparative method for DNA extraction from most types of storage card. 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.
- 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.
- forensicGEM™ extracted DNA is largely single-stranded because of the heat step. If double-stranded DNA is required, the 95° step can be omitted and standard downstream purification procedures can be used.
- For accurate yield assessment, a qPCR is recommended. The DNA produced by forensicGEM™ 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.
- forensicGEM™ is stable for 30 days at 4º C, for longer-term storage forensicGEM™ should be stored at -20ºC.
- For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C
Typical Results
DNA Profile
Figure 1: Typical AmpFlSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems) profile from a buccal swab of a female subject. 25 µl of extract was used in a standard Identifiler PCR. CR.