FTA Punch

 

  Typical Results

 


Figure 1: Typical AmpFlSTR® Identifiler™ kit (Applied Biosystems) profile from an FTA stored buccal swab of a female subject.


 

SCS qPCR


Figure 2: Taq-Man real-time PCR trace of DNA extracted from saliva samples collected and stored on Whatman® FTA® Indicating Micro Cards using the prepGEM Storage Card Saliva kit.. Standards (5.5 ng/μl; 1.85 ng/μl; 0.620 ng/μl; 0.210 ng/μl; 0.068 ng/μl and 0.023 ng/μl) are shown in red. Samples (blue). DNA extractions performed on five saliva samples that had been stored on Whatman® FTA® Indicating Micro Cards. Extractions were performed on 2mm diameter punches according to the standard ZyGEM protocols as detailed in the relevant quick-start guide. Each PCR was performed in duplicate, with 5 μl of extracted DNA being used as a template in a final reaction volume of 25 μl.

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. Remove one or two 2 mm discs from the card-stored sample and place into a thin-walled PCR tube or a 96-well tray.

2. Wash the disk in 100 µl of DNA-free water by incubating at room temperature for 15 minutes. Aspirate the water from the disc and discard.

Uneven application of the swab onto the storage card results in DNA yield variations. For the best results, punch in the center of the area where the sample was applied. 

SCS Workflow

Extraction Method

1. Add:

• 4 µl of 10x buffer SKY BLUE
• 35 µl of DNA-free water
• 1 µl forensicGEM

2. Incubate at 75°C for 5 minutes.
3. Incubate at 95°C for 2 minutes. A thermal cycler can be used for this step
4. Pipette the solution to a new tube.

The DNA is in this solution - not the punch.

 

Technical Tips

  • The ZyGEM Storage Card procedure can be automated using most liquid handling robots.
  • ZyGEM Storage Card is a preparative method for DNA extraction from most types of storage card. Its purpose is to lyse cells and to strip the DNA of nucleoproteins. Extracted DNA can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
  • Storage cards for cells and DNA contain preservatives that can seriously inhibit Taq DNA polymerase. The pre-soak step is to remove these inhibitors and is essential for reliable results.
  • ZyGEM 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 downstream purification procedures can be used.
  • Yields can vary significantly. This is not a result of the extraction method, but due to the uneven distribution of cellular material on the card. If you find the yields are too low, you can add moore punches.
  • For accurate yield assessment, a qPCR is recommended. The yield of DNA from storage cards is too low for any other method.
  • 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 card punch.
  • forensicGEM / prepGEM is stable for 30 days at 4ºC, for longer-term storage it should be stored at -20ºC.
  • For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.

 

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