Hi Katherine

If your primers worked before and the only difference is that you're using a different extraction kit it may be that the extraction kit is faulty. This does happen and usually the company will replace it if it's a manufacturing fault.  Or perhaps the filter used in the new kit to capture DNA is not fine enough and you're losing template during isolation?

That said, repeated freezing and thawing of primers can cause them to degrade, have you tried a fresh batch?

Alternatively, if this is a different water sample it may be that whatever eDNA you're trying to amplify with your current primer set is not present in the new template for some reason. Non-specific bands below your band of interest usually means you need to up your annealing temperature but may also mean that your primers have degraded and are now binding nonspecifically.

Also for eDNA, smaller bands may represent specificity to another organism with similar sequence not present in your initial sample?

Hope you sort it out. Good luck.

Regards,

Nathaniel

@Nathaniel

I think the kits are not faulty because I've used them on other samples and they worked properly (not ATL samples - another set of samples also taken from water). Others have used the same kit as me at the same time and theirs work perfectly. If anything, it's either my primers (but they still work really well with tissue sample) or for some reason the Atlantic salmon in the tank did not give me ATL DNA at all on my filters.

No idea! I've also tried a new batch of primers. same results.

Hi Katherine,

The problem lies in the PCR reaction that has not worked. The issue of primers can be ruled out if you included a positive control that always worked in all the optimizations you did. Have you tried to run PCR with fresh primers made out of stock solution? Are you able to quantify the DNA to have an idea of the amount of DNA you add in your PCR reaction? Because the amount of total DNA in a PCR does affect the outcome of a PCR.

@Jacques

My PCR positive controls always worked even when I changed the conditions many times. The DNA quantity was low but it has always been low when extracted from water samples however I have always managed to get bands using other primers or ATL samples taken from the same tank last year (except I extracted them using PCI last year and not kits). I have used new primers (reordered them). These samples will work with other fish primers...just not my apecieis specific ones even though my species spexific ones will work on other ATL water samples. 

So the only difference this year was I used a kit which gave me better quality DNA (still same low quantity) and I get about 50ul worth. 

It's a very puzzling situation. Let's hope for more insight to that issue.

I think you should try some other annealing tempretures

I propose you to do a gradient PCR.

So I've been trying different gradients, dilutions, new reagents etc. I still cannot get any bands to show up during the first PCR...it will only show up once I run the PCR product from the first run, and onto a second run.

I've done numerous touchdown PCRs with different settings, and once it appeared I got very faint faint bands - once I did a replicate of the whole process I got nothing. 

I no longer know what to do...my positives work every time and my negatives are always clear. I just need the bands to come up during the first PCR run. 

Did you ever manage to solve this issue Katherine? We are having a similar problem at the moment..

Thanks, Alex