I am trying to extract DNA from an unknown specimen, most likely animal fat. I have used a silica column-based kit (Invitrogen) with no luck. Although the specimen is highly processed, I am hoping I can get lucky.
I have used QIAGEN kits to extract DNA and RNA from fresh/frozen adipose tissues of mice and rats, but in any case, large amounts of lipids interfere with the extraction yields. Therefore, I first frozed the tissue and then crushed it. After suspending the tissue in lysis buffer, the lipid was separated by centrifugation and cooling before adding alcohol.
This step was very important.
I would suggest that as little lipids as possible be carried over to the next step.
By the way, what process did the specimen undergo? The answer depends on that as well.
Good luck!
Yasuhiro Nishida
Thanks. I am not sure how it was processed as it is a confiscation, and we want to do species ID. The end result was a big hard block. I assume the animal fat was boiled at some point. Would adding Triton X-100 to the digestion buffer help?
I see. It seems to me that the extraction difficulty is high.
It's hard to say because boiling or Triton-X 100 can work better or worse depending on the timing and occasion.
I don't know what the block is, but does it look like the specimen is inside what looks like white to amber plastic/resin?
If so, the specimen is most likely embedded in paraffin.
For example, DNA can be extracted (though not completely) from formalin-fixed and/or paraffin-embedded samples, and depending on the quality of the DNA, amplicon sequencing may also be possible from it.
*DNA extraction from paraffin embedded sample
Article DNA Extraction from Paraffin Embedded Material for Genetic a...
You can also purchase kits to produce this at QIAGEN.
If this is the case, you need to start by making sections and removing the wax-like resin (paraffin) from the block with xylene.
The xylene is then removed with alcohol and the extraction process is similar to normal DNA extraction.
Perhaps the samples are often amplified by PCR and then sequenced by classical sequencer such as ABI PRISM 3100, but that may not give a good result.
I suggest using NGS with amplicon sequencing to increase the depth of sequencing to get more accurate data.
Article Performance of amplicon-based next generation DNA sequencing...
Article Assessment of the quality of DNA from various formalin-fixed...
Article Using FFPE Tissue in Genomic Analyses: Advantages, Disadvant...
Good luck!
Yasuhiro Nishida
*General tips of DNA extraction
https://www.promega.co.uk/resources/guides/nucleic-acid-analysis/dna-purification/?cs=y
Article Impact of storage conditions on the quality of nucleic acids...
- Badges
- Science topic