Hello Annelise,

Pls clear some point

First one- You have isolated DNA by QIAGEN DNEasy Blood & tissue Kit

final elution in which Elution buffer or sterile water

initial DNA storage temp ?

any freeze-thaw condition

Hello Kamlesh,

I did the final elution with Buffer AE, as specified in the protocol.

Initial DNA storage temp was 4°C for the DNA isolated in 2018, and -20°C for the DNA isolated this year, and both have been thawed maximum of 5 times, definitely not more than that.

should not be a problem. what was your dna concentration? initial and purity (260/280) ? what is your PCR product length ? long or short? use more template DNA or increase number of cycles to 35. usually store DNA at -20C for long term storage only short term 4C... -20 C samples should be good for PCR for a long time

Thanks Jaroslaw, DNA concentrations are between 35 to 110ng/µl and purity is all around the 1.80 mark. PCR product ranges between 450bp and 1000bp (one piece was 6kb, as we were working with an unkown piece, but I have that 6kb piece already cloned and am working with that separately now). Cycles are already at 35, and most storage has been at -20°C

so its not very high but fair enough :) 6kb is long 450-1kb are normal size and should work with diluted DNA . for degradation you can always run it on a gel and see a main band or smear.

good luck

if you are happy that you followed the dna extraction process accurately then you might want to try re ordering the primers particularly if your primers are dissolved in water not TE as degraded primers will also cause pcr to fail. If you are after a specific amplimer then nested pcr often works well.

You should better check the DNA concentration again. Moreover, after thawing and before using in PCR step, you can follow a gentle vortex and a quick centrifuge of you experimental sample.

Did you include positive PCR controls in your experiments? did they work properly? Degradation shouldn't be (normally) an issue.

Thanks for all the comments.

I ran all my extractions on a gel, to see if we could see a smear or not, and most of them looked very nice with no smear.

Positive controls, I have made primers for GAPDH in pigeon myself, they have worked just after the extraction, but later has not worked anymore, on multiple PCRS.

I have checked with others in my lab regarding good PCR and sample handling practice, and they have advised me I have been handling them correctly and following appropriate procedure.

Now the question, why would the PCR not be working, when I am following proper practice (and being very careful), and it had worked just after the extraction? Others in my lab group have been using the same Taq Polymerase and other parts of the kit, and they are having no problems with their PCRs

Had you optimize annealing temperature of the primer that you are using ?

Your positive control reaction must be run in the same experiment as your samples. The control must be a template that you know for sure that works properly with the oligos your are using.

Sometimes that cannot be done. If this is your case, and provided you thoroughly checked your procedures, I advice you to look at DNA quality (see for example Article The peril of PCR inhibitors in environmental samples: the ca...

And yes, as pointed out above optimizing annealing temperature is usually helpful to.

Hope this helps.