DNA collection kit Oragene® FAQ

Collection

What is the DNA yield?

From a study of 208 samples, the median amount of DNA was 110 ug per 2 mL of saliva, and the average was 100 ug. The 25% percentile was 62 ug and the 75% percentile was 158 ug. The range was 10 to 375 ug. Click here for the complete whitepaper.

What is the best way to quantify my DNA yield?

We recommend quantifying your DNA with a fluorescent dye such as SYBR Green or PicoGreen (Molecular Probes) because these dyes bind to double-stranded DNA. Quantification by absorbance will tend to over-estimate the amount of DNA because of absorbance by RNA co-purified with the DNA. For more details, please consult our quantification protocols DNA Quantification using SYBR Green I Dye and Micro-Plate Reader.)

The median yield of my Oragene•DNA samples is lower than 110 ug. Why is this?

There is a degree of variability in the amount of DNA in saliva. Even in the same donor, the amount of DNA in saliva can vary from day to day. Additionally, the total DNA yield can vary depending on the method of purification used. The amount of DNA found in some Oragene DNA/saliva samples may exceed the binding capacity of some column and bead-based purification kits; in these cases, not all the DNA in the sample will be recovered and the median yields will appear to be lower. Typically, the manual method of purification will recover all of the DNA in the sample.

What can I do to increase the yield of DNA?

Low yields often result from samples containing less than 2 mL of saliva. To optimize DNA yields ensure your donors spit the full 2 mL of saliva as indicated in the user instructions. Click here for the complete whitepaper.

Does the Oragene•DNA kit still work if someone spits more or less than 2 mL of saliva?

Yes, the Oragene•DNA chemistry can handle variability in saliva volumes from 1.5 to 2.5 mL.

How can I collect saliva from infants and young children who cannot spit?

Please consult our protocol for "Using Saliva Sponges to Collect DNA samples from Infants & Young Children" for more information.

How much bacterial DNA is in my Oragene•DNA samples?

Using a real-time PCR assay with bacterial 16S rRNA primers, the median amount of bacterial DNA was 11.8%. In comparison, DNA collected with mouthwash contains about 50% bacterial DNA and buccal swabs may be almost 90% bacterial. The small amount of bacterial DNA content in Oragene•DNA does not affect downstream applications. Click here for the complete whitepaper.

Storage

What temperature should I store my saliva samples in Oragene•DNA?

Oragene•DNA stabilizes the DNA in saliva across a range of temperatures. Saliva samples may be stored at room temperature or frozen at temperatures such as -20°C or -80°C, depending on which is most convenient for you. Please note that storage at 4°C is not recommended for Oragene samples. Consult our "DNA Stability with Oragene•DNA" application note for more details.

Can saliva samples in Oragene•DNA frozen and then thawed?

Yes, saliva samples may undergo at least 3 freeze-thaw cycles with no evidence of DNA degradation. Please consult our "Long-term Storage Recommendations" application note for more details.

Purification

The A260/280 ratio of my purified DNA is low. What can I do to improve this?

DNA from saliva purified with the Oragene•DNA ethanol precipitation protocol should have A260/280 ratios ranging from 1.6 to 1.9. If your ratio is low, try the following trouble-shooting tips:

(1) check that the Oragene•DNA samples have been incubated for at least 1 hour at 50°C before purification,
(2) when purifying the Oragene•DNA sample, be sure not to disturb the protein pellet after the first centrifugation step,
(3) check that your spectrophotometer is subtracting the A320 value from the A260 and A280 values (A320 represents the background turbidity of the sample), and
(4) check that the individual A260 and A280 values are in the range from 0.1 to 1.0 (i.e. within the linear range of the spectrophotometer).

Please see our white paper entitled "From turbidity to clarity: Simple methods to improve the A260/A280 ratio of Oragene®•DNA purified DNA samples" for more information.

When I run an agarose gel on my purified DNA, there is a large smear at the bottom of the gel. Is this degraded DNA?

No, the smear is low molecular weight RNA.

How can I remove the RNA from my samples?

Please consult our protocol "RNA removal by double-RNase digestion" for more information.

Do we need to incubate samples for an hour at 50°C if using other sample extraction methods than what is offered by DNA Genotek?

Yes, every Oragene sample must be incubated at 50°C for a minimum of 1 hour in a waterbath or 2 hours in a dry incubator prior to DNA extraction. This ensures that cell lysis is complete and that the sample is homogenous.

Downstream applications

Can Whole Genome Amplification (WGA) be performed on DNA from Oragene•DNA?

Yes, DNA from Oragene•DNA can be used as the template for commercially-available WGA kits such as GenomiPhi (GE Healthcare), GenomePlex (Sigma-Aldrich), and REPLI-g (Qiagen).

Does the bacterial DNA in Oragene•DNA samples interfere with downstream applications?

DNA from Oragene•DNA performs the same as DNA from blood for downstream applications because the amount of bacterial DNA in Oragene•DNA samples is low, and PCR-based technologies have good specificity. For your interest, our application note entitled "SNP genotyping of Oragene•DNA™ with SNP stream®" shows that the SNP genotyping calls are identical with DNA from blood versus DNA from Oragene•DNA. Additionally, Affymetrix has published a technical note showing that the performance of DNA from Oragene•DNA/saliva samples is identical to that of DNA from blood in SNP microarray applications.

Do you have any recommendations for using DNA from Oragene•DNA with Affymetrix or Illumina chips?

Yes, for Affymetrix or Illumina chips, we recommend purifying the Oragene•DNA samples using the Oragene Manual Purification Method with the following modifications:
1. Dissolve the final DNA pellet in 50 uL of water or TE buffer instead of the 100 uL suggested in the protocol. This will help ensure that the DNA concentration is suitable for use on microarrays.
Additionally, the purified DNA MUST be quantified using a fluorescent method such as Sybr Green or Picogreen. See our DNA Quantification protocols for more information.