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DNA Extraction
Using
prepGEM™ Insect
prepGEM™ Insect is a formulation specifically developed for extracting quality DNA from whole insects or insect parts. It yields PCR ready DNA, which shows reduced inhibition from various compounds that have been shown to limit PCR performance. The extracted DNA is suitable for barcoding, sequencing and SNP applications.
- The kit uses optimized methods and formulations to provide DNA in 20 minutes
- Reactions are closed-tube and hands-off.
- All buffers and reagents are PCR compatible, so no downstream purification is required.
These features make prepGEM™ Insect ideal for large-scale automation in environmental screening programs. The few steps and simple methodology of prepGEM™ Insect allow easy adaptation to most liquid handling platforms for full or partial automation. This allows for full walk away capability or parallel processing of thousands of samples per day with off-deck incubation.
For more information on this product, please review:
prepGEM™ Insec Application Note
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Workflow
Extraction Method
The following procedure outlines general guidelines. Different sample types will require slight method variations.
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 forceps, scalpels and dissection surfaces in 0.05% hypochlorite bleach. Rinse thoroughly with DNA-free water.
1. Insects or parts of insects should be placed in a thin-walled PCR tube for extraction. Approximately 1-2 mm of total tissue should be used.
2. Add to the material:
- 35 µl of PCR -grade water
- 4 µl of 10x buffer BLACK
- 1 ul prepGEM™
3. Incubate at 75 °C for 15 minutes.
4. Incubate at 95°C for 5 - 15 minutes.
5. Centrifuge 2 minutes at 13,000 x g if required.
Typically, the supernatant from fresh insects should be diluted 1:10 in water and 1 µl used in a 25 µl PCR. For older or degraded samples, undiluted extracts can be used and the volume increased.
Notes:
1: For dried insects, it is advisable to break up the exoskeleton with forceps and to pre-soak the tissue overnight at 4°C in 40 µl of 1x buffer 3. The following day, add 1 µl of prepGEM™ and proceed to Step 3.
2: For very small insects, these volumes can be reduced further.
3: There will be little apparent reduction in the amount of visible material. The purpose of the step is to lyse the cells in order to extract DNA and to strip the DNA of nucleoproteins. Excessive digestion will release more potential inhibitors into the solution.
4: 5 minutes is sufficient in most cases.
5: Centrifugation is undesirable for automation and should not be necessary.
Technical Tips
- The prepGEM™ Insect procedure can be automated using most liquid handling robots.
- prepGEM™ Insect is a preparative method for DNA extraction - it is not a purification protocol. Its purpose is to lyse cells and to strip the DNA of nucleoproteins. The formulation creates DNA that can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
- prepGEM™ 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. DNA produced by prepGEM™ is approximately 90% single-stranded. If standard fluorescent chelating dyes are to be used for quantification, then 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 sample after extraction, aspirate the supernatant when cool and store separately from the tissue.
- Depending on the type of insects present in a sample and their state of desiccation, grinding and/or re-hydration may be required to achieve optimal yield.
- The use of EDTA in the sample-handling protocol can reduce enzyme performance in the extraction. This problem can be overcome by adding CaCl2 to a final concentration of 200 µM.
- prepGEM™ is stable for 30 days at 4º C; for longer-term storage prepGEM™ should be stored at -20ºC.
- For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.