Saliva Quantifiler

Figure 1: Blue: Examples of Quantifiler® traces from forensicGEM™ buccal swab extracts. Four extractions are shown. Red: Standards generated using the manufacturer's DNA. Green: Internal control. Calculated concentrations are between 2.5 and 2.3 ng / µl for the replicates


Saliva yields

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


Identifiler saliva

Figure 3: Typical AmpFlSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems) profile from a buccal swab of a male subject. 2 µl of extract was used in a standard Identifiler PCR.

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.


Method

Wash the buccal swab in the minimum amount of DNA-free water to cover the swab. Typically, a cotton swab requires 300-400 µl. Use a rolling action against the tube sides and squeeze the swab against the tube to remove as much of the liquid as possible.

Method workflow saliva

1. In a thin-walled PCR tube add (agitate suspension prior to adding the following):

  • 69 µl water
  • 20 µl of the swab eluate
  • 10 µl of 10x buffer BLUE
  • 1 µl forensicGEM

2. Incubate at 75°C for 10 minutes.
3. Incubate at 95°C for 2 minutes. A thermal cycler can be used for this step

The sample is now ready for quantification and analysis.


Technical Tips

  • The prepGEM / forensicGEM Saliva procedures can be automated using most liquid handling robots.
  • ZyGEM Saliva is a preparative method for DNA extraction - it is not a purification protocol.  Its purpose it to lyse cells and strip the DNA of nucleoproteins. The formulation creates DNA that can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
  • There is no concentration step in the procedure and so the final concentration is dependent on: 1) The vigor with which the swab was taken. 2) The type of swab. 3) The volume of buffer used to wash the swab. We recommend using the smallest amount of buffer required when washing the swab in order to maximize the DNA concentration.
  • prepGEM / forensicGEM 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 purification procedures can be used.
  • For accurate yield assessment, a qPCR is recommended.  The DNA produced by these kits is approximately 90% single-stranded. If standard fluorescent chelating dyes are to be used for quantification, this factor should be taken into consideration. For best results, prepare and manage samples at 4ºC, or on ice, before and after extraction. See our Application Note on how to quantify DNA using fluorescent dyes. 
  • The  prepGEM / forensicGEM enzyme is stable at room temperature, however longer-term storage and to safeguard against contamination, we recommend that you store the tube at -20ºC.
  • For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.

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