VacutainersQuantifler Blood

Figure 1: Blue: Examples of Quantifiler® traces from forensicGEM™ blood extracts. Samples from four individuals were used. Red: Standards generated using the manufacture's DNA


Blood yields

Figure 2: Yields from blood samples (four individuals) using forensicGEM™ and other extraction methods. Error bars are one standard deviation.


Figure 3: 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.

This method is recommended for ZyGEM DNA extraction directly from liquid 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.


Work area 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 with 0.05% hypochlorite bleach. Rinse thoroughly with DNA-free water. 

Method

Blood method workflow

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 2 minutes*.

3. Incubate at 95°C for 5 minutes. A thermal cycler can be used for these steps.

*See the Application note for optimising your extractions with other blood volumes.

4. Centrifuge in a microcentrifuge at full speed for 5 minutes. 
5. Aspirate supernatant. Typically, around 0.5 ng / µl is expected for fresh blood. 


Technical Tips

  • The ZyGEM Blood procedure can be automated using most liquid handling robots.
  • The prepGEM / 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.
  • It doesn't matter that the DNA has some residual pink colour. If you follow the usage guidelines (1 µl extract in a 25 µl PCR) you will have no problems.
  • 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.
  • The enzymes is stable for at room temperature but we recommend storing the tube at -20ºC once it has been opened. This is to protect it from accidental contamination.
  • For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.

Downloadable PDF files