Hi, everyone.
I'm using the "Qiagen DNeasy Blood & Tissue Kit" to extract genomic DNA from insects (beetle, Coleoptera) and facing hard trouble now.
The problem as follow.
Size of gDNA of my subject taxon is abnormal in the result from "Genomic DNA screentape assay" as attached image. I tested my target beetles and other groups (six other families) as 'negative control' to comparing the result, however, only my target sample was weird. All other group beetles showed a perfect peak in the range of 16000~18000 bp, while my target beetle group had no proper peak. I think that DNA degradation occurred.
- All tests were processed with other group beetles (negative control) in same condition, but negative control group's result were perfect. I think that experiment condition is not consideration.
- All my target samples were certainly fresh. They were just dead before experiment. I think that freshness of samples is not matter, too.
Here is my protocol for DNA extraction, that mostly refer the manual from Qiagen. I also note with italic font that some control that I tried (but it was not work).
1) Grinding the sample in 1.5ml tube
- It was not small species, about 5mm size.
- I tried to grind samples with two option, whole body & excluding abdomen.
2) Lysis process in incubator
- I made premix of "Buffer ATL (9) + Proteinase K (1)" before experiment.
- Put the premix (200µl) into grinded sample
- Base condition was '56℃ / 450 rpm vortexing'
- I tried to control amount of Proteinase K (10, 20, 30µl in 200µl volume)
- I tried to control incubating condition: temperature (40, 50, 56, 60℃), time (24, 8, 4, 2h), rpm (450, off).
3) After incubating, put Buffer AL (200µl)
- Incubating additional 10 minutes in same condition until gel-form product (produce after put Buffer AL) was dissolved.
4) Put EtOH (200µl) into sample
- briefly vortexing.
5) Move supernatant into spin-column and centrifuge the column (8000rpm / 1min)
- Change bottom tube after finsh the centrifuging.
7) Put Buffer AW1 (500µl) and centrifuge the column (8000rpm / 1min)
- Change bottom tube after finsh the centrifuging.
- I tried to centrifuge once again with same condition to certainly dry.
8) Put Buffer AW2 (500µl) and centrifuge the column (14000rpm / 3min)
- Change bottom tube with sterilized 1.5ml tube for storing DNA after finsh the centrifuging.
- I tried to centrifuge once again with same condition to certainly dry.
9) Put Buffer AE (110µl) and centrifuge the column (8000rpm / 1min)
- I tried to elute DNA twice (60µl at first, 50µl at second time)
- I tried to preheat Buffer AE in 56℃ incubator
If you can find some advisable points, it will be great help for me.
2 possible factors are enzyme degradation. When you lyse the cells enzymes are release that eat dna. The addition of EDTA removes divalent salts that allow these enzymes to work so lysing cold and with EDTA helps create longer dna and protect it until the proteins have been purified away
Rough handling so mechanical breaking of the long strands of dna can happen with grinding of samples and with shaking of dna containing solutions or with the use of benchtop whirlimix type shakers
In addition, freezing & thawing cycles will shear DNA that has been purified.
Also, why are you adding in so many variables? Don't you have a standard protocol for the proteinase K amount & incubation time & temperature?
Unless your goal is to optimize the DNA extraction procedure for long DNA molecules, it's just a tool to give you DNA.
Good luck!
Thanks Paul,
Firstly, thanks for sincere advices.
Can I know some manual or publication as reference to add EDTA? Maybe it will be solution.
About mechanical breaking of DNA, I don't agree. Other negative control groups (all of them are beetle groups) that tested simultaneously showed perfect results. Also, I tested with many variation (not vortexing in incubator / non-grinded sample), but it didn't work.
Thanks Katie,
In this case, freezing & thawing cycles matter is not a issue. The used target samples were dead in ETOH or naturally just before experiment. Freezing & thawing was not happen. But thanks for advice.
Of course, I have standard protocol based on Qiagen manual. There was not a single problem previous experiments for other beetles. But for this time, it didn't work, so I tried to improve it with application of many variations. But it doesn't work yet...
Maniatis and Frisch or some similar manual will have a reference to using edta but the attached link may expand on the usage
https://aboutthecharacter.com/qa/what-is-the-function-of-edta-in-dna-extraction.html
Well done if you are getting long dna it can be difficult. In the early days of pulsed field gel electrophoresis it was so hard to get long dna that the cells were suspended in agarose and the dna extracted in situ and enzyme digested in situ to avoid mechanical fracture
Read my research virus is living and its treatment whole answer is given their
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