Oragene FAQs
Collection
- Q: What is the DNA yield?
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A: 208From 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.
- Q: Is RNA included in the reported DNA yield?
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A: 110µg is a number, measured with fluorescent pigment, which is why RNA is not included. According to the absorption spectrometer's measurement method, as long as RNase is not treated, DNA+RNA can be measured. It is believed, there is equal quantity of RNA and DNA, which is why it is guessed, that from 2ml saliva sample can be extracted 200µg nucleic acid(DNA+ RNA).
- Q: What is the best way to quantify my DNA yield?
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A: 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 protocol.
- Q: The median yield of my Oragene samples is lower than 110µg . Why is this?
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A: 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.
- Q: What can I do to increase the yield of DNA?
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A: DNA yield is proportional to the volume of saliva that is collected. To get more DNA, ask donors to spit more saliva. Although the standard Oragene instructions ask donors to spit 2 mL of saliva, the Oragene chemistry can preserve up to 4 mL of saliva. Click here for the complete whitepaper.
- Q: Does the Oragene kit still work if someone spits less than 2 mL of saliva?
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A: Yes, the Oragene chemistry can preserve a wide range of saliva volumes - from microliters up to 4 mL of saliva. Click here for the complete whitepaper.
- Q: What is the source of the DNA in saliva?
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A: The source of DNA is buccal epithelial cells and white blood cells.
- Q: How can I collect saliva from infants and young children who cannot spit?
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A: Please consult our protocol for "Using Saliva Sponges to Collect DNA samples from Infants & Young Children" for more information.)
- Q: How can I collect saliva from dogs, cows, horses, and other animals?
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A: Please consult our protocol for "Using Saliva Sponges to Collect DNA samples from Infants & Young Children" for more information on how to collect saliva from humans or animals that are unable to spit.
- Q: How much bacterial DNA is in my Oragene samples?
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A: Using a real-time PCR assay with bacterial 16S rRNA primers, the median amount of bacterial DNA was 6.8%, with a range from 2 to 29%. 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 does not affect downstream applications. Click here for the complete whitepaper.)
- Q: Do you have any recommendations on how to conduct a mailing study with the Oragene kits?
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A: Yes, please consult our Mailing Recommendations.
- Q: There is a yellow-colored substance at the bottom of the Oragene vessel. What is it?
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A: It is an important substance, which preserves the DNA in the saliva. Please, do not take it away.
Storage
- Q: What temperature should I store my saliva samples in Oragene?
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A: The Oragene DNA-preserving solution stabilizes the DNA in saliva across a range of temperatures. Saliva samples may be stored at room temperature, 4℃ in the refrigerator, or frozen at temperatures such as -20℃ or -80℃, depending on which is most convenient for you. Please consult our "DNA Stability with Oragene" application note for more details.
- Q: Can saliva samples in Oragene frozen and then thawed?
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A: Yes, saliva samples may undergo at least five freeze-thaw cycles with no evidence of DNA degradation. Please consult our "Long-term Storage Recommendations" application note for more details.
Purification
- Q: When is the appropriate time for the incubation step before the purification to take place?
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A: Right after the saliva collection and right before the purification: find a suitable for you moment to do it within this period.
- Q: What happens at the incubation step?
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A: It takes place, in order to extract enough DNA and keep nuclease's inactivation permanent.
- Q: What happens at the step, where purification liquid is added?
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A: If purification liquid is added, protein, mucin and cell fragments will be precipitated and removed
- Q: What happens at the step, where ethanol is added?
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A: According to the adding of ethanol, DNA can become insoluble. According to precipitation, it will take out DNA, rather than liquid.
*Oragene purification protocol is a modified precipitation method, using the usual salting out and ethanol. - Q: Is it possible to decrease the 10min on ice, used for incubation?
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A: Yes, the time can be shortened, but 10min are calculated to be the best period for keeping the best purification condition and extract max DNA quantity. If the time is shortened, it may be difficult to achieve the standard level
- Q: Why do you put equal quantity of ethanol and supernatant and not double the ethanol, compared to the supernatant's quantity, when you have the step with adding ethanol?
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A: If glycogen is added, indeed precipitation is possible. However, during the last purification, the DNA will contain glycogen and this may influence the 260/280 level of turbidity. Still, if your goal is to achieve just something close to normal PCR analysis, then there is no problem to add glycogen.
- Q: After you add ethanol, there is a step, where you should leave it for 10min at room temperature. Can't you simplify the process by adding glycogen?
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A: If glycogen is added, indeed precipitation is possible. However, during the last purification, the DNA will contain glycogen and this may influence the 260/280 level of turbidity. Still, if your goal is to achieve just something close to normal PCR analysis, then there is no problem to add glycogen.
- Q: Aren't 2min for centrifuge separation too short?
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A: The longer the time of centrifuge separation is, the bigger volume of collected DNA can be expected. However, comparing to the spent time, the collected volume is really paltry. We wanted to shorten the time of the protocol as much as possible and 2min was the period, which would not influence the volume of collected DNA so much.
- Q: The A260/280 ratio of my purified DNA is low. What can I do to improve this?
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A: DNA from saliva purified with the Oragene 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 samples have been incubated for at least 1 hour at 50℃ before purification(2)When purifying the Oragene 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)(4)Check that the individual A260 and A280 values are in the range from 0.1 to 1.0
- Q: Can saliva samples stored in Oragene be purified using methods other than the ethanol precipitation protocol supplied with the kit
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A: Yes, homebrew methods such as phenol/chloroform extraction or commercial kits such as QIAamp (Qiagen) and PUREGENE (Gentra) may be used to purify DNA from Oragene samples.
- Q: Can saliva samples stored in Oragene be purified using automated DNA extraction robots?
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A: Yes, DNA from Oragene samples have been purified using robots such as the BioRobot EZ1 (Qiagen), AutoPure (Gentra), Magtration 12GC (PSS Bio), MagNA Pure LC (Roche), among others.
- Q: 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?
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A: No, the smear is low molecular weight
- Q: How can I remove the RNA from my samples?
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A: Please consult our "RNA removal by double-RNase digestion" for more information.
- Q: How to storage the purified DNA?
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A: Dissolved in TE buffer at 4 degrees room temperature for 1-2 months; for long period storage, we recommend preserving at minus 20 degrees. (Data file)
- Q: How long is the shortest possible period of time between collecting the saliva and the purification?
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A: About 100min
Downstream applications
- Q: Which downstream applications are compatible with DNA from Oragene?
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A: DNA from Oragene is fully functional with PCR, real-time PCR, multiplex PCR, SNP genotyping, microarrays, sequencing, RFLP, etc.
- Q: Can Whole Genome Amplification (WGA) be performed on DNA from Oragene?
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A: Yes, DNA from Oragene can be used as the template for commercially-available WGA kits such as GenomiPhi (GE Healthcare) , GenomePlex (Sigma-Aldrich) , and REPLI-g (Qiagen).
- Q: Does the bacterial DNA in Oragene samples interfere with downstream applications?
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A: DNA from Oragene performs the same as DNA from blood for downstream applications because the amount of bacterial DNA in Oragene samples is low, and PCR-based technologies have good specificity. For your interest, our application note entitled "SNP genotyping of Oragene with SNP stream" shows that the SNP genotyping calls are identical with DNA from blood versus DNA from Oragene.
DNASelf-Collection Kit
Oragene®-DNA
TEL)+81-044-853-2958 (Did)
FAX)+81-044-854-1979
KYODO INTERNATIONAL INC.
2-10-9 Miyazaki,
Miyamae-ku, Kawasaki-shi,
Kanagawa-ken, 216-0033,
Japan
FAX)+81-044-854-1979
2-10-9 Miyazaki,
Miyamae-ku, Kawasaki-shi,
Kanagawa-ken, 216-0033,
Japan
