MicroGEM ForensicGEM Sperm DNA Extraction Kits for Use with Thermal Cyclers User Guide

MicroGEM ForensicGEM Sperm DNA Extraction Kits for Use with Thermal

Cyclers

This Quick Start Guide provides methods to extract DNA from a range of sample types.

Principle of the Extraction

MicroGEM extraction products use a unique mixture of thermophilic and mesophilic enzymes. Protocols involve a 75oC step to activate MicroGEM’s thermophilic proteinase to lyse cells, denature nucleases, and strip DNA of nucleosomes. Certain protocols in this Quick Start Guide include an initial low-temperature step when mesophilic enzymes ae required. A final 95ºC step deactivates the thermophilic proteinase resulting in analytics-ready DNA.

Kit Components and Storage Reagents

Acrosolv is a mixture of reagents that weaken tissue cell walls. It is delivered as a lyophilized powder. This should be resuspended in DNA-free water as follows:

Reagent storage: forensicGEM reagents are delivered at room temperature but on arrival should be stored at
4ºC. After tubes have been opened, the forensicGEM enzyme should be placed at -20ºC. Once Acrosolv has been rehydrated, it is stable for 7-12 months at -20ºC. If you do not plan to use all of the Acrosolv immediately, it is recommended that you aliquot Acrosolv into smaller volumes and store at -20ºC immediately after rehydration. The buffers can remain at 4ºC for convenience.

MicroGEM Reagent QC

Our reagents are made from certified DNA-free chemicals and solutions and all buffers and enzymes are treated with DNAse and UV before shipment.

General Instructions, Precautions and Technical Tips

• All manipulations should be performed in a clean room or a PCR hood.
• Labcoats, gloves and hairnets should be worn at all times.
• Use only certified DNA-free tubes and reagents.
• Wash equipment that will come into contact with the sample in 0.05% bleach. Rinse thoroughly with DNA-free water.
• forensicGEM is a preparative method for DNA extraction. The method lyses cells and removes nucleoproteins from the DNA. Extracted DNA can be used for many types of genotyping including SNP and STR analysis as well as a quantitative, multiplex and end-point PCR.
• There is no concentration step in the procedure and so the concentration of the extract is dependent on: 1) The quality of the sample; 2) In the case of swabs, the type of swab and the volume of water used to wash the swab; 3) The extraction volume.
• DNA extracted using forensicGEM is largely single-stranded because of the 95ºC heat step. For accurate yield assessment, a qPCR is recommended. If standard fluorescent chelating dyes are to be used for normalizing samples, then we recommend taking a sample of the extract before the 95ºC step. Alternatively, you can generate a standard curve using a previously-made extract that has been quantified. As with any preparative method for nucleic acid extraction, best results are obtained when samples are handled at 4ºC, or on ice, before and after extraction.
• For long term storage of the extracted DNA, add TE buffer and store at -20ºC.

Sample Preparation and Extraction Procedures

Semen
Stains and Swabs
The processing of the sample will vary dependent on the sample type. For liquid samples, try to keep the volume of the liquid below 10 µl. With cotton swabs, add ¼ of the swab directly to the extraction cocktail. Stained fabric can be swabbed or small portions added directly.

1. Place or pipette the sample into a thin-walled PCR tube.

2. Add:
   10 µl 10x ORANGE+ Buffer
   2 µl forensicGEM
   10 µl Acrosolv
   Water to 100 µl
   

3. Mix the sample by vortexing.

4. In a thermal cycler, incubate:
   52ºC for 5 minutes
   75ºC for 3 minutes

5. Aspirate the extract away from any residual material.
   The DNA is in this solution. Do not discard.

Troubleshooting

For troubleshooting, please see our guide, “MicroGEM Method Optimization and Troubleshooting” using the following link:
https://www.microgembio.com/m_resource/microgem-method-optimisation-and- troubleshooting/

Additional Information
For publications/app notes on your specific sample type, please visit:

https://www.microgembio.com/resources/

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At MicroGEM, our goal is to democratize molecular biology, enabling a broader spectrum of users to both employ and benefit from molecular techniques. The first step is the simplification of sample preparation. Our temperature- driven, single-tube process simplifies and reduces the number of steps for traditional nucleic acid extraction, resulting in high-quality extracts with reduced contamination and high yields – all in minutes, not hours.

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All trademarks are the property of their respective owners; forensicGEM, phytoGEM, prepGEM, RNAGEM, and MicroGEM are trademarks in use by the MicroGEM group.
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