Hi Antara,

I am not sure what you try to amligfy and what your template is (maybe you can give some more information on your reaction, e.g the what template and how many of the template you are using, what primer concetration do you use and the annealing temperature of your primer?). This would make trouble shooting easier.

However, when your PCR worked once and is not working anymore it is likely that one of you components is not working anymore.

My first guess ist that your template might be degraded. Check the interity of your template on a gel. Do not use to much of your template as this could also make problems (for a plasmid 10 ng should be ok, for genomic DNA around 50 ng).

However, you could also have problems with other components of your reaction - like primers or polymerase - so use fresh batches of all your PCR components if possibel.

Regarding your primer dimers - try lower primer concentration and /or higher annealing temperatures. Increasing the denaturation time can also help and you should also consider Hot start PCR.

Hope this helps

My GOI is about 6 kb, and i am using 1 µl each of forward and reverse primer and DNA conc. between 20 and 30 ng/ml. I have put a lot of PCR according to the instruction of Phusion polymerase enzyme but didn't get any good amplification

I would suggest the following: Immediate action: Run a positive control (with known working template/primers) and a negative control (NTC) with your current setup. This will tell you if the problem is with your reagents/cycler or specifically with your target's amplification. Also, include your problematic samples.

Focus on Reagents: If controls fail, replace all reagents (especially enzyme, dNTPs, and water) with fresh, reliable stocks.

Optimize Annealing Temperature: If controls work, the problem is likely specific to your reaction. Set up a gradient PCR to find the optimal annealing temperature, starting higher than your previous optimal to try and eliminate primer dimers.

Primer Review: Re-evaluate your primer design using an online tool. If extensive troubleshooting fails, consider designing new primers. Hot Start: If not using already, try a hot-start polymerase.

Hi Antara,

sorry that it took me sometime before I come back to you. First I think Phusion polymerase is a good choice for amplifying large amplicons. However, if you have these large amplicons you really have to have a good template - did you check the integrity on gel in the meantime (would be interesting to see)?

As Abdulwahid M. Dhahir mentioned is always good to have a positive control to see if other PCRs on the same template are working. A negative control is also a good idea (however this is to exclude false positives it this does't seem to be your problem in the moment). For a posistive control I would recommend an amplicon which is not to small, because if your template integrity is not good, small amplicons might be still amplified and you can get still problems which larger ones.

I am not sure about your template concentration - you wrote 20-30 ng/ml. This might be a typo and you mean 20-30 ng/µl. If your concentration is really 20-30 ng/ml this would mean that you have a concentration of 0,02-0,03 ng/µl and even if you put 36,5 µl of this template in a 50 µl reaction (what is the maximum for the Phusion-Pol per Protocol and I would not recommend doing this) you would have only 0,7 - 1,1 ng template in your rection. This would be not enough, especially if you have genomic DNA as a template (see above). So please double check your template concentration.

For your primer concentration I would need do know the concentration of your primers or the end concentration of the primers in your reaction to get an idea if your primer concentration is ok.

To high and to low primer concentration could both interfere with your PCR (final concentration should be 0,1-1 µM ( this would mean 0,05 µl to 0,5 µl of a 100pmol/µl stock - what is often used).

Optimizing primer concetration and annealing temperature (gradient PCR might be helpful) is often not easy, but key for a sucessfull PCR.

If you get at least a faint band of your GOI you might also increase the cycle number - especially with low template concentration - however overcycling might lead to smearing and I would recommend to increase your template concentration (usually 33-35 cycles should be enough)

.

I would be really interested in seeing your initial amplification (which worked) and a gel where you checked you template (measuring the DNA concentration with photometer is not enough as you will not see if the DNA is intact or degraded). Maybe I could get some additional ideas from this.

I hope this helps a little bit

KR Dörthe

Sudden failure of PCR that was working usually means that your primers have degraded. Get a fresh aliquot and see if that helps!

Good luck

The possibilities are numerous, such as the primers not hybridizing well, the template DNA being insufficient, or the conditions used being restrictive, among others. My suggestion is that you evaluate various parameters, such as DNA concentration, temperature gradients, and MgCl2 concentrations, among others.