DNA degradation issue

[WendyJiang]

Hi, I have a problem with our DNA sample.
Our DNA sample were extracted by Trizol from human blood. We store DNA stock in -80. We need to shift some DNA aliquotes by Fedex to our collaborators. So we took out 200ug of DNA, checked DNA quality, and then stored them in -20 freezer for 1 or 2 weeks and shipped out.
Amazingly, our colloborator lab checked the DNA quality and found most of them were degraded (Gel picture attached). The 260/280 is around ~1.6 and there were DNA obviously stuck in the loading holes.
Any one can give me a clue what could happen to our sample? Any solution to solve this problem?

Really appreciate it!

: )

[nucacidhunter]

I wonder if you checked DNA in gel after extraction or taking out from -80C freezer or before shipment. Quantification is not enough for DNA QC. A full QC would include quantification with dsDNA binding dye such as PicoGreen, a gel or microfluidic device run to look for size distribution and NanoDrop or similar device to look for absorption at 260, 230 and 280 nm wavelength. Possible reasons:

1- DNA was degraded before or during extraction. You can check this with aliquots that you still may have in your lab.
2- Degradation during transit, it would depend on transit time, temperature and purity
3- Streaking DNA in wells is sign of impurity and viscous material carry over which indicates that the extraction procedure was non-optimal

[WendyJiang]

Hi, thank you very much for your suggestion.
Yes, we did check DNA in gel when we take it out from -80 and the quality is very good. After that, we aliquoted and get them shipped.
I think you are right, our extraction may not be optimal.
We use Trizol to extract buffet coat from blood sample.
Somehow, DNA degraded during shipment... : (


Is there any kit we should use to purify our DNA sample?
What buffer we should use to preserve DNA to protect them from degradation during transit?

Thank you so much for your help! Really appreciate it!

: )

[nucacidhunter]

Best buffer to minimise DNA degradation is TE, but check to make sure it is compatible with downstream application (EDTA in TE buffer can affect activity of some enzymes). Shipping in ice is recommended if the transit time is long. For overnight shipment even RT is OK. I would also suggest looking at following web page:
http://www.ogt.co.uk/resources/liter...ge_and_quality

I never have used Trizol for DNA extraction. I hope someone can suggest a suitable kit for your extraction.

[WendyJiang]

Hi Nucacidhunter,
The benifit of using Trizol is that we can get DNA/RNA/Protein at same time with high quantity. The advantage should be impurity for each component.
I think TE buffer will help in this case. I will also try to purify DNA by kit from Qiagen.
After I try those steps, hope the problem can be solved.

: )

[nucacidhunter]

You can do a column or bead based clean-up of DNA after extraction to remove impurities if you are happy with quality and quantity of RNA and protein from your samples using Trizol.