Hi all,
I am trying to create an in-house storage buffer to include in our saliva collection tubes for future DNA isolation. Ideally our lab is looking for alternatives to the typical DNA Genotek Oragene kits - we'd like the samples to be able to sit at room temperature prior to analysis. It's obviously proven very difficult to determine what exactly is in the Oragene storage buffer as well as the subsequent PrepIT L2P reagent, but I am thinking that the storage buffer contains all steps through cell lysis, and the PrepIT contains protease.
Any suggestions/words of wisdom would be much appreciated.
So far, I'm thinking of a Tris-HCl / EDTA /NaOH / SDS / RNase / (glucose?) concoction for the storage buffer...
Hi Kathleen, the best storage conditions for saliva, if you could, is storing it straight at -80oC.
RT storage of saliva will be problematic because of the bacteria and enzymes in the samples. Storage at -80oC will diminish all hydrolase activities and bacterial growth.
Hi Steingrimur Stefansson , thank you very much for your response! Unfortunately I'm planning a project during which participants will need to collect multiple samples in their own home. I've had success storing samples at room temperature with the DNA Genotek kits, whose proprietary storage buffer accounts for bacterial growth, but we'd like to try to figure out a solution ourselves.
Hi Kathleen, you could try collecting saliva 1:1 into 50% ethanol.
Steingrimur Stefansson, thank you for the help, I will look into this!
Kathleen Krol Was this helpful? I am conducting an immunoassay research on oral fluid samples and my protocol requires that I store aliquot of these sample for future study, how do you advise I do this, because my collection device does not come with buffer or transport media in it? Steingrimur Stefansson
Hi Tolulope Oginni , I'm still working out the kinks but so far I've been able to isolate DNA from saliva samples stored for at least a week in a Tris-HCl EDTA buffer with SDS (still figuring out percentage, but so far I've used .1% and it worked). Though the DNA is fragmented on a gel, the yield has been far greater when I freeze the samples at -20 as opposed to storing them at room temp. This is definitely a work in progress, though. I'm not sure what you need for your immunoassay project, but my buffer is essentially a cell lysis so I can get to the DNA. If you don't want to lyse the cells, don't include the SDS!
Hi Kathleen, during my PhD thesis, I have collected cheek epithelial cells from 1014 elderly individuals by using swabs. We kept the swabs in the lysis buffer at room temperature until DNA extraction, which we did 2 months later. The lysis buffer was composed of 50 mM Tris (pH 8.0), 50 mM Na2EDTA (pH 7.4), 50 mM Sucrose, 100 mM NaCl and 1 % SDS.
Hi Mehmet Baki Yokes , it's really helpful to hear others' experiences, so thank you for sharing! I'm starting to get to a buffer that works for me, which looks quite similar to yours. My interest is in downstream DNA methylation analyses, so what's been key for me is creating a buffer that not only gives me high quality DNA, but that also performs well during pyrosequencing. Thanks again for your feedback on this!
I'am acctualty try the same in my master thesis. I had good DNA integrity for over 3 weeks with 95% Ethanol and also with a lysis buffer instead of Oragene.
I also tryed Tris-HCl/EDTA/SDS but had high degradations.
What did you use instead of PrepIT L2P?
Hi Felix Holocher , I'm using a potassium acetate solution in place of PrepIT.
Hey Kathleen Krol ,
thanks for your answer. Did you get the same results like prepIT?
Literature says 0,1-0,5M potassium acetate, which concentrations did you used?
Potassium acetate solution (for 2 mL stock):
· 1.2 mL 5M potassium acetate
· 0.23 mL glacial acetic acid
· 0.57 mL DNase/RNase free H20
I add 20ul potassium acetate solution into 500ul aliquots of sample (saliva + lysis buffer).
Hi,
Dear @ Kathleen Krol, the final buffer you mentioned are useful for lysis or preservation of saliva DNA?
Hi,
Dear @Kathleen Krol, the final buffer you mentioned are useful for lysis or preservation of saliva DNA?
Hi Maryam Daneshpour ,
I used the Lysis buffer for both lysis and storage. But do not know its components.
Hi Maryam Daneshpour , the potassium acetate solution I mentioned was used as a protein precipitation solution for DNA isolation, not storage (can work in place of DNA Genotek's PrepIT-L2P. I used a lysis buffer for storage, so yes - the storage buffer will lyse the cells! I will note that I still haven't determined the best lysis solution for my needs (downstream DNA methylation). I've currently switched back to the Oragene kits until I can really trust myself. :)
Hi Kathleen Krol , did you figure out the percentage of buffer solution? can you share it if you can. i need it too.. thank you..
Hi Mustafa yıldız , unfortunately I wasn't able to sort out a buffer that provided me with DNA worthy of downstream pyrosequencing. I suppose it depends on what you're using the DNA for. I was able to isolate DNA using a 1% SDS buffer, sat at room temp for 4 and 7 days. The quality seemed fine on a gel but for whatever reason did not perform well in pyro. It's possible you could still use the DNA for genotyping (or methods with less room for error), but I had very high replicate mean deviation in triplicate DNA methylation analyses, which is my main variable of interest.
Hello, we are doing studies in epigenetics and we wanted to know if it is only stored with SDS or with another substance. Also if you have observed good quality when storing the samples at -20 or 4°C for methylation analysis. Thank you!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA