Sample Workflow for Blood Kits

Extraction Method
This method is recommended for DNA extraction directly from liquid human 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.

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 forensicGEM™
• 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 forensicGEM™ Blood procedure can be automated using most liquid handling robots.
• forensicGEM™ 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 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 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.
  
• 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: The results below were generated from an extraction from 2.5 µl of fresh blood. 1 µl of the extract was profiled using AmpFlSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems). A. forensicGEM™  extracted DNA. B. Manufacturer supplied control DNA. C. No forensicGEM™ added to extraction

Yield of DNA

Table 1: The yields were calculated for forensicGEM™ using both a blot-based system, ABI Quantiblot® and a qPCR method, Quantifiler®. Sample size = 2.5 µl b lood. Final Volume forensicGEM™ extraction = 100 µl

• QIAGEN® reports typical yields of 3 µg of DNA from 2 ml of blood (2.5 x 105 l eukocytes per ml) for their QIAamp® columns. This equates to a yield of 1.5 ng DNA / µl of blood.
• Chemagen® reports yields of 60-120 µg of DNA from 3 ml of blood. This equates to a total yield of 20 – 40 ng DNA / µl of blood.

Quantifiler® Traces

Figure 2: Blue: Examples of Quantifiler® traces from forensicGEM™ blood extracts. Samples from four individuals were used. Red: Standards generated using the manufacture's DNA

Yield of DNA

Figure 3: Yields from blood samples (four individuals) using forensicGEM™ and other extraction methods. Error bars are one standard deviation.

Notes:

• The phenol method used 20 µl of blood treated with proteinase K in a 200 µl volume. Samples were extracted with phenol, phenol+chloroform+IAA (25:24:1) and chloroform. Samples were precipitated in ethanol and then resuspended on 50 µl of TE buffer.
• All other methods were scaled variants of the manufacturer's instructions.