How about Dntps? Did you change it? Also, ddh20 you need to check as well. Try to use original pcr set up for troubleshooting . Do not change a lot of parameters once. I think if you go step by step you will fix your problem. Need to have negative and positive controls as well.

Good luck.

Ali

Thank you, I just made new dNTPs so I will try that next

There can be many reasons for this problems.

* May be the PCR itself is not working, so try the same reaction on other PCR.

* To check the quality of primer, mix forward and reverse primers (5 microliter each) and run it on 1 per cent gel.

* Check properly the annealing temperature

* Annealing temperature varies with the Taq enzyme used

* Extension time which you are keeping should match the speed of Taq enzyme and time should be properly calculated based on amplicon length.

* Compatibility of buffer and Taq should match.

* PCR components s/a dNTPs, MQ (autoclaved), primers should be of good quality.

* Concentration of template should not be too low, and if it is low, increase the number of cycles.

I may have left some other answers, hopefully it may help you.

You can try keeping gradient PCR as well first because maybe primer is not able to bind at the kept temperature

Hello Alexandra Margolin

I am so sorry that your gels and PCR stoped working.

Have you run any gels lately that worked? Have you tried re-running any leftovers from a previous PCR that worked?

I would recommend to make sure that you do not have issues with your electrophoresis before you start teasing the PCR reaction. Make sure your run buffer is fresh, your DNA dye is working, and you use the appropriate loading buffer. Finally make sure your gel is running and you see bubbles durping electrophoresis.

If your electrophoresis works, then I would start teasing the PCR reaction, foll the advice that was given earlier.

hope it works soon.

best of luck.

Olga

Hi,

I guess the problem is probably with the template DNA… if it hasn't work even after changing the dNTPs.

When did you use the template DNA last time and how was it stored? As you have only checked it with nanodrop, doesn't ensure that your template is intact. Many times nanodrop is not so accurate, moreover it will give you the concentration of DNA even it's fragmented. Do check your template on gel once and keep one positive control.

GOOD LUCK!

Hello. It's better that you perform a standard pcr with some kit. Just to check if your basic components are working. This would restore your confidence too.... Happy pcr to you. 👍

what is the product length of your PCR product and what % of gel are you running? If you are using EtBr then check its condition also.

problem may also be their!!

Ali Akgul made a sensible suggestion. Do not go changing lots of parameters otherwise you will end up being confused.

Check your template not only by nanodrop but on agarose gel as this will visually tell you if it is intact.

Clean apparatus and all gel equipment (including glass vessel used to melt agar). Do not use old TEA buffers, make fresh stock.

Other easy things as suggested: dNTPS (these should be aliquoted as they do not like repeated F/T), Fresh sterile water.

If you have a nuclease problem then your PCR may appear as a smear on agarose. Check to see if apparatus/agarose runs well by just running out a plasmid or any DNA of known size. If ok then there is nothing wrong with the gel running and it is a PCR issue.

Are you sure it is actually a problem with PCR and not the gel electrophoresis? Re-run an old PCR reaction as positive control.

Check the quality of the template DNA by gel electrophoresis BTW. Smeared or faint bands suggest degradation.

Hi Alexandra,

it seems you already tried the "usual suspects". Several years ago we had a similar case - PCR stopped working, borrowed and bought all the components anew, re-isolated DNA... In the end it turned out that our de-ionized water had gone bad (some infection in the outlet!). Maybe you could retry your PCR with water from a different source, just in case.

Good luck,

Klaus

I have already had the same problem and it was the dNTPs.

Hi Leticia,

yes, from my experience that is the first component "going bad". So, if I have to trouble shoot PCR - that's the first test. Since my first encountering this problem I diligently aliquote the dNTPs to avoid multiple freeze/thaw cycles :-)

Klaus

Try changing the Nuclease free water which you use for PCR reactions

Presumably you run DNA size standards on every gel? In which case, if you see those your gel then electrophoresis is not the problem.

Sometimes small bands don't show up on agarose gels because ethidium (and equivalents like Sybr Safe) run in the opposite direction to DNA (i.e. they run up the gel towards your black electrode). That can mean that the bottom end of your gel lacks an intercalating agent and so DNA there doesn't show up. To counteract that you can include ethidium or equivalent in your running buffer during the run, so that it enters the gel to replace that migrating up through it. Use the same concentration that you would when making your gel.

If you do think it is an electrophoretic detection problem then you could always perform PCR (including controls + & - polymerase), then carry out a cleanup step, and detect DNA via nanodrop (or similar). The control + polymerase and your sample ought to give you a much higher reading for DNA concentration than the control with no polymerase (which should only have template and possibly primers captured during the cleanup step).

I think it's unlikely to be the electrophoresis though. More likely the PCR, in which case primers, dNTPs and nuclease free water are the obvious things to check, and you have loads of good advise on that above - but you must get a positive control from somwhere! Ask around the department - even do the PCR in someone else's lab (can often change things!).

It may be a good way to check the sample in gel rather than Nanodrop.

Because some degradation of sample may not be found in Nanodrop.

Good luck.

Hi Alexandra,

First of all, crosscheck all the reagents you are using for PCR. Secondly, the EtBr, because it plays the most important role in this process. Then see the concentration of your stock Template DNA and find out the working concentration of the template. If everything is in the right place, then optimize the PCR condition. Don't reduce or increase the concentration of every reagent you are using for PCR. Go ahead with one by one.

Good Luck with your PCR

Checking the pH of your buffers might solve the issue with the gels.

To be sure that the problem is at the level of PCR manipulation, try to migrate amplifiers that have already worked before.

if bands are visible at this moment the problem is at the level of the PCR otherwise it is the electrophoresis. try to work with the same concentrations as at the beginning and do not change the parameters of your PCR. If the DNA extracts are of good quality, and the dNtps are diluted in sterile water, and you were not wrong in the dilution of the primers then the problem is maybe at the level of the electrophoresis, so try to work with a TAE trampon freshly prepared, the ideal is to work with the TBE, adjusting the pH of the migration buffer is also important.

Alexandra Margolin

Hi did you find the solution to your problem? I am currently facing the same problem and I don't know what to do. My pcr was working fine and now suddenly am not getting any bands. Same template, same protocol, same machines... am not sure i understand what has happened and it has been driving me crazy for the past 2 weeks :( Please help.

Hi Depika, did you try fresh dNTPs, already?

Klaus Eimert

yes I have.

Hi Depika,

let's try to pinpoint the problem. Do other primers work with the same setup? Do fresh DNA isolates work? Can you borrow fresh pcr components (including water!) to troubleshoot?

Cheers,

Klaus

Alongside what people already shared, have you tried also checking electricity infrastructure out and inside machines as well? Amperage can also be the issue if the voltage of the electricity is unstable. Also, DNA binding color might also be the issue, being degraded. I had that issue, purchasing the color of the company which was degraded and of very bad quality. Also, see if the samples are somehow different - maybe you haven't extracted DNA from samples and Nano-drop is registrating some other diluted substance. I had this challenge, when i realize that i actually didn't have any DNA in some of the samples coming from organisms from another environment. When you identify the issue, please, share it with us and how did you solve the problem. Good luck!

Depika Prasad I set up an experimental procedure that would let me know if there was a problem with my template DNA or if it was my reagents that were giving me trouble. I made one master mix from my reagents and one master mix with someone else's reagents. I took a positive control from a previous PCR reaction and diluted it 1:100 four times with water (or you can use TE buffer). This gave me a very small fraction of dilute, "good and working" DNA template that I added to my master mix and then put in one PCR tube. I then used the original DNA template and added that to another PCR tube with my master mix. Then I mixed this diluted positive control template with my original DNA template and put that into a third tube with my master mix. I did this procedure twice (one for each master mix). I then ran the gel electrophoresis. If you were to get bands on only the positive control lane, then you know it is your DNA template that is faulty. I got bands on the master mix that I made out of another person's reagents, so I concluded that one of my reagents had gone bad. Hope this helps and good luck!

There are many factors that AFFECTING YOUR ISSUE, IN MY OPINION, IT IS BETTER TO RUN A negative and positive control to check all your materials

Good luck