PCR problems are usually due to bad template, bad primers, or bad PCR/melting point conditions. Do other PCRs work? If you reordered primers and made a new template (did you R. digest the plasmid to make sure its good quality DNA, UV spec?).

If you miniprep your bacteria instead of doing a colony PCR does this solve your problem? Colony PCR is really quick but can contaminate your bench with DNAses, bacteria, agar which can mess up PCRs. Might be a bad batch of competent cells, same thing happened to me with a Us11 plasmid. I went through all the same troubleshooting and eventually everything just went back to normal, all i can hypothesize was that we ran out of the bad bacteria and changed to a new lot.

Dilute your heated sample to 1/10. Then use 1-5 ul for PCR.

Hi Marc-Antoine,

1. Check how much bacteria you're inputing into your colony PCR. I've run into the issue before where I put too much gDNA and ended up with really fuzzy or no bands at all. I'm assuming that you're using a pipette tip to pick your bacteria so it is unlikely that you are putting too much but that is one thing you can check.

2. Check your annealing temperature. You can do this by running a gradient PCR.

3. In addition to all the factors you have listed above, you can try even try checking your polymerase to see if it has degraded. Maybe someone in the lab took it out for too long or didn't keep it in an icebox.

4. If all the above does not work, sometimes it is easier to re-transform your bacteria and try a new set of materials. Unfortunately, colony PCR can be very difficult to troubleshoot even though it seems like a simple experiment.

Best~!

Check how much bacteria you're inputing into your colony PCR. I've run into the issue before where I put too much gDNA and ended up with really fuzzy or no bands at all. I'm assuming that you're using a pipette tip to pick your bacteria so it is unlikely that you are putting too much but that is one thing you can check. Check your annealing temperature. You can do this by running a gradient PCR. In addition to all the factors you have listed above, you can try even try checking your polymerase to see if it has degraded. Maybe someone in the lab took it out for too long or didn't keep it in an icebox.

How many cycles do you use for your PCR reaction? Do you have a clear, and specific band on the gel? If the PCR results have clear and specific band, it may be the PCR is working besides low yields. Increasing the number of cycles might improve the yields.

Do all the bands appear weak, or only these specific colony PCRs?  Does your ladder still look OK?  Wait...you tried different agarose and buffer, didn't you?  Yes, I see that now.  It does sound like you have too much DNA (like mentioned above).  I think I would try a dilution first.  Also perhaps trouble-shoot your thermal cycler.  It is still running at the correct temperatures?  Nothing suspicious there?

Okay it was all about the gel red. With winter and cold bench i stored it at RT and it seems it had some precipitations that vortexing wasnt able to break.

I heated it at 55°C for 2min and it worked perfectly. So it was indeed the gel red that was the problem.

Thanks for all your answers. Appreciate that.

Doing colony PCR of E.Coli is pretty easy, suspend your colony in 20 ul water, heat it for 5 minutes at 99C. Centrifuge at 5000 rpm for 5min. Use 5ul as template and use the same things as you use before, just raise your annealing temperature 1-2 degrees. I hope you'll get your required results. 

I suspect you are using too much DNA for PCR. Can you qubit your DNA and dilute accordingly for PCR then it should work well.