Hi,

Did you check the concentration of the plasmid DNA after elution from paper. Probably, concentration of plasmid DNA in 0.5ul is not enough to visualise on the agarose gel. If it is too low, I would recommend you to transform the plasmid (2-3ul from eluted supernatant) in bacteria and plate with appropriate antibiotics. Then, perhaps proceed with colony PCR and plasmid extraction from the confirmed clones.

In case the above transformation did not work, you could try to concentrate the plasmid in sup. using Speedvac and bring the sup. to smaller volume. Then transform the bacteria again to get some success.

l would still keep the filter paper till you finish all these steps and you may need re-elute again from paper if nothing works. I had some issues eluting plasmids from filter paper. Hope this helps.

Cheers,

Godwin

Basically do exactly what Godwin says.

I think you did correct to extract it, as you probably are aware you need enough water or buffer that will still actually be able to be retrieved from the filter paper and not just remain in it, I used to use anywhere from 30-50 uL. I used to let the filter spot soak in the buffer (I cut out the filter circle and put it into a eppendorf tube) at room temp for a couple hours and then came back to it later to collect supernatant and perform nanodrop, unfortunately I have no idea if that is better or worse than 37c for 3 minutes, but I have never had issues extracting plasmid dna from filter paper.

Thank you for your suggestions. Will try the transformation and update.