The simple methodology of prepGEM™ Blood allows technicians at all levels to produce large numbers of quality DNA samples in just minutes

DNA Extraction Using prepGEM™ Blood

prepGEM™ Blood is a simple enzymatic DNA extraction system specifically optimised to overcome the PCR inhibition commonly found with single tube extraction protocols for blood. It is ideal for high throughput genotyping applications, PCR applications, or SNP detection: whenever rapid results are required.

The kit uses optimized methods and formulations to provide DNA in 30 minutes.
There are no multiple centrifugation steps, washes, vacuums, or columns.
Reactions are closed-tube and hands-off.
All buffers and reagents are PCR compatible, so no downstream purification is required.

These features make prepGEM™ Blood ideal for large-scale automation in medical research workflows. The few steps and simple methodology of prepGEM™ Blood 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.

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Workflow

Extraction Method

This method is recommended for DNA extraction directly from whole animal blood. Incubations can be performed either in a thermal cycler, water bath, or using an automated robotic workstation fitted with Peltier temperature-controlled heating blocks.

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. In a thin-walled PCR tube add:

86.5 µl of PCR grade water
10 µl of 10x buffer RED
2.5 µl of liquid blood
1 µl prepGEM™

For EDTA stored blood, an additional 2 µl of 10 mM CaCl2 should be added PURPLE

2. Incubate at 75°C for 15 minutes.
3. Incubate at 95°C for 15 minutes. A thermal cycler can be used for this step
4. Centrifuge in a microcentrifuge at full speed for 2 minutes.
5. Aspirate supernatant.

Typically, around 0.5 ng/ µl should be expected for fresh blood.

Technical Tips

The prepGEM™ Blood procedure can be automated using most liquid handling robots.
prepGEM™ Blood 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.
For accurate yield assessment, a qPCR is recommended. The DNA produced by prepGEM™ 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 precipitated residue and store separately.
If extracting DNA from blood containing EDTA, CaCl2 should be added to a final concentration of 200 µM to improve the enzyme activity.
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.