Dear Colleagues, is it necessary to freeze cephalopods samples for DNA extraction already preserved in Ethanol 96% ? I used LCO-HCO primers without success to loliginid samples, whilst morphologically these present specimens looked the same as previous samples being successfully primered by the same sequence of LCO-HCO. Could that be because the specimens were wildtype ones? Also, which one should we trust more to prove the presence of template DNA please? Nannodrop or elphor? Thanks.
I don't think you should freeze the samples unless you need to store them long-term. I suggest preserving them in 96%-ethanol and store them in the fridge (-4C). If they were stored at room temperature PCR might not work. You may try to play with PCR settings (annealing temp, extension time, etc.) to see if the primers will work, there are also might be specific primers for your species and universal LCO_HCO might not be ideal. I used nanodrop, and it worked for me (for genomic DNA) - for checking the presence of PCR products I suggest gel separation. Hope that might help! Best of luck!
Dear Alina, thanks a lot for your sharing. I leave my whole batch in room temp for ca. 2 weeks due to lack of space in the fridge. Now it is safe though. I will let you know whether the neglected samples give me proper results still.
Thanks..best of luck to you too.
I hope 2 weeks shouldn't be a big problem. Thank you :-) Good luck!
Thank you Hasan, I meant my samples were neglected outside the fridge for ca. 2 weeks and I hope it is still ok. BTW, what temperature do you use (more or less) in freezer? Also, could we still extract DNA from samples preserved in formalin please? I tried without success. Cheers.
Freezing (-20 or -80 Degree)
is effective to get qualitative nd quantitative DNA.
My dear Colleagues, further explanation of my problem about checking DNA template: after the extraction I put the extract in nd, the OD 260/280 is between 1 to 1.39. Then l recheck it further by elphor and it comes that nothing in it, just scratch. Why was that please, since the elphor of its amplicon is also empty. For the latest, that could be due to primer incompatibility mentioned by Alina..
You may want to run a gel and see if there is something... The gel picture might be helpful to check if there is a problem with primers, or DNA degradation, or something else... Hope that would work!
I might be wrong, but yes, to me it looks like there is no DNA template there..
I do not think you were wrong, because both nd and gel may assist in informing about the presence of DNA, but just why in this case (l repeated several in time extraction using chelex, several master mix and proteinase-K) the nd did not match the gel. And further amplification using universal primer (previously successful) showed nothing in the amplicon by gel examination. Thanks a lot Alina for tour thought and sharing..
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