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Article by: Scott Rabuka

7 Simple Steps to Maximize DNA Yield with Oragene•DNA

2010-02-19

At DNA Genotek, we understand the importance of high quality DNA samples to support our customers' research and clinical studies. As a result, we work with our customers to ensure the results they obtain with our products are optimized. Implementing proven techniques and procedures for maximizing the DNA yield from Oragene/saliva samples can increase the yield and improve the efficiency of laboratory processes and quality of downstream results.  Recently, we created a list of 'best practices' to ensure that you achieve the maximum possible yield from Oragene/saliva samples. We want to share this list with you.

Follow these tips and tricks to maximize the DNA yield from Oragene 2 ml saliva samples.

  1. Collect the required volume of saliva. The required volume of saliva is 2 mL. Collecting less than the required volume will result in lower yields.  Saliva may sometimes be foamy, so it is important to ensure that liquid saliva, not the bubbles, reaches the 'fill-to' line.
  2. Follow the instructions on the Oragene package carefully. This includes not eating, drinking, smoking, chewing gum or brushing your teeth for 30 minutes prior to giving the saliva sample.
  3. Finish spitting within 30 minutes. The full saliva sample should be collected within 30 minutes and the Oragene tube or vial should be capped immediately. Waiting longer than 30 minutes may decrease the yield and quality of the DNA.  A 2 ml saliva collection should typically take less than 5 minutes.
  4. Take an aliquot for DNA extraction after incubation at 50°C. Saliva is quite viscous and can trap DNA if not processed correctly. The 50°C incubation step releases DNA from the Oragene/saliva sample and reduces the viscosity, producing a homogenous sample. Taking an aliquot prior to the incubation step may give a lower DNA yield and may provide inconsistent results when multiple aliquots are purified from the same sample.
  5. Add the correct amount of alcohol to precipitate the DNA. It is important to add an equal volume of room-temperature ethanol (95-100%) to the clear supernatant after the centrifugation step. For example, 500 µL of ethanol should be mixed with 500 µL of supernatant. The aqueous phase should be mixed gently and thoroughly with the ethanol to ensure complete precipitation of DNA. A visible clot of DNA will usually be seen as the ethanol mixes with the aqueous phase. Adding more than 1 volume of ethanol and/or using cold ethanol will not improve DNA recovery and may increase the amount of impurities that precipitate.
  6. Allow a sufficient period of time to rehydrate the DNA. DNA extracted from Oragene is high molecular weight and may take some time to dissolve completely in buffer. The rehydration solution should be a low salt buffer such as TE. We recommend overnight incubation at room temperature to ensure complete rehydration of the DNA. For best results, the final concentration of DNA should be less than 200 µg/mL (200 ng/µL). Drying the sample after ethanol precipitation is not recommended because this can significantly increase the rehydration time. The rehydration time may be shortened by incubating at 50°C for 1 hour with occasional vortexing. Once the DNA is dissolved, you should briefly vortex or pipet the sample several times prior to taking an aliquot.
    NOTE: If higher concentrations are required, you may rehydrate the DNA pellet with as little as 50 µl of TE buffer.
  7. Quantifying the DNA. It is important to remember that Oragene collects about 2 mL of saliva and mixes it with 2 mL of Oragene solution. Therefore, the total volume of Oragene/saliva solution is about 4 mL. The total DNA yield for the 4 mL should be > 20 µg. We recommend quantifying DNA by fluorimetry using a fluorescent dye such as SYBR Green.  Quantification by this method is more accurate than absorbance.

We hope these 7 steps will help you maximize the DNA yield from all your Oragene samples.

Request free trial kits of any DNA Genotek saliva kit.

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