I am using a Qiagen DNA kit-DNeasy Blood and tissue kit- and would like to know if an overnight incubation in the initial lysis step can negatively affect the DNA.
Hello Maggie Reddy,
I think it doesn´t, but you should read it in your kit´s handbook. I utilised BioSprint 15 DNA Kit (Qiagen) and ChargeSwitch gDNA Micro Tissue Kit (Invitrogen) and I didn´t observe any negative effect. But I insist you should consult the technical handbook of your kit.
Hope this helps. Good luck with your DNA extractions!!!
Thanks a lot Fernando. Yes, according to the kit's protocol a 1-3 hour incubation should be sufficient and if the tissue is hardy (as is lobster muscle tissue) then an over night incubation might be neccessary. I just wanted to check if extended exposure to the lysis buffer and proteinase K could negatively affect the DNA in any way.
Hi again, Maggie,
According with the protocol of the BioSprint 15 DNA Kit (tissues protocol): "Lysis time varies depending on the type of tissue processed. Lysis is usually complete in 1-3 h. If it is more convenient, samples can be lysed overnight; this will not affect the DNA quality". It refers to the QIAGEN Proteinase K, but I guess all the proteinases work similarly, so I think 12-14 hours doesn´t hurt the DNA.
Only for satisfying my curiosity ( :) ):
- Which part of the lobster body do you utilise for the DNA extraction?
- Do you remove the hard-calcified parts of the tissue? When I extracted DNA from beetles, I used 1-2 legs and I used to grind the tissue in order to make more easy the penetration of the proteinase K., but other people use muscles directly removed from the torax. I think it could be easy to extract muscles from a leg of a lobster, couldn´t it?
Best,
Fernando.
Hi Fernado
Thanks again. I extract DNA from the muscle tissue so I break apart the tough shell and finely mince the tissue.
Hi Maggie,
I extracted a lot of tissue samples with the Qiagen Kit. Tissue from paraffin embedded and frozen tissue. I always had an incubation with proteinase K over night and this was never a problem for subsequent PCR or sequencing.
I wish you all the best for your extractions.
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