I am not being unable to isolate DNA from J2 stage nematodes. The nematodes have been extracted from gall and currently are in water. I used extraction methods using sonnicator, glass beads, mortar pestle using liquid nitrogen and even qiagen blood and tissue DNA isolation kit, but no luck so far. I must be doing something wrong somewhere. I will be highly obliged if someone can share a working protocol for DNA isolation from juvenile nematodes or some tips how I can achieve this. Thanks
Hi!
You can filtrate your water with nematodes through a membrane filter to concentrate nematodes and after it perform DNA isolation with DNeasy PowerWater Kit
what makes you think that there is no DNA?
Agarose gel needs over 20ng to show dna and you may have less than this if there is not much starting material. Have you measured by spectrometer or tried doing a PCR to see if you do have some dna purified?
Hi,
I think qiagen blood and tissue DNA isolation kit is okay for DNA extraction from any tissue, as I used it for hard tissue and got good result. You can check DNA quantity by using Dr. Paul Rutland suggestion, I'll prefer to go for spectrometer by NanoDrop; because PCR result depend on primer and protocol selection. After confirmation, if you still find that there is no DNA then check your lysis buffer quality or add extra lysis enzyme to get better result while extracting.
Thanks
Thank you everyone for your comments and specially Mr. Paul Rutland. I will certainly follow your suggestions. I havent yet quantified the DNA as it didnt produce any band. I will do it now.
Hi Nirjhar,
I would like to add one more point. Make sure you use protenase K to degrade proteins because if your sample is rich in protein it tangles with DNA and make gel which makes the isolation difficult.
Hi Nirjhar,
You can follow the protocol described by Yan et al., 2008
Article Detection and Discrimination of Pratylenchus neglectus and P...
"Ten nematodes of mixed juvenile and adult stages were handpicked using a dental pick, put into 20 µl of sterilized nanopure water on a concave glass slide, and cut into two pieces under a dissecting microscope. Then, 10 µl of suspension containing nematode pieces were pipetted into a 0.2-ml sterile Eppendorf tube with 8 µl of lysis buffer (500 mM KCl; 100 mM Tris-Cl, pH 8.3; 15 mM MgCl2; 10 mM dithiothreitol [DTT]; 4.5% Tween 20; and 0.1% gelatin). The tube contents were frozen at –20°C for at least 20 min, then thawed, and 2 µl of proteinase K at 600 µg/ml was added. The tubes were incubated at 65°C in a water bath for 1 h, and consecutively at 95°C for 10 min to inactivate proteinase K. The nematode lysis mix was centrifuged at 16,000 × g for 5 min and the supernatant was transferred to a new 0.2-ml tube and stored in –20°C until used as the DNA template."
Mihai
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