First check the primer sequences to make sure they are correct.

next, it may be simply a question of changing the annealing temperature for your new primer set. You say you are using the same conditions as for a previous target, but do not say that you optimised the annealing temperatures of either. 

You may also need to check the primer concentrations and adjust those. 

Finally, you may need to adjust the PCR reaction conditions. We have started to use KAPA's master mix, which is both fast and cheap. For SYBR green assays up to 200 bp, we use 1 sec denaturation, 1 sec polymerisation, for Hydrolysis probes and amplicons >200 we increase the polymerisation time to 2 seconds.

Hi Sailesh,

When the primer and amplicon changes, there could be a lot of difference in the PCR efficiency and its now a new story altogether. It could be due to high GC content of the template, primer dimers, different primer Tm etc. Consider yourself  lucky to have some product, you could also have ended up having nothing. Try subtle changes in conditions (Tm, Mg2+ concn, DMSO etc.) and hope you get a better yield. Good Luck

Hello Sailesh,

As already mentioned by Manoj and Setphen, you need to optimize PCR for each target gene and primer pair differently. So, just to start, check the following:

1) Template DNA: whether it is genomic DNA or a plasmid from where you are amplifying, that will decide the initial template you take.

2) Primers: The Tm of primers is of utmost importance, whether you are taking the correct annealing temperature, whether the primers are specific and do you see a specific product, do you see primer dimers, in that case, decrease the primer concentration. These are a few things you can check easily.

3) dNTP concentration and stocks, are they working well.

4) Polymease: The reaction can vary even when you have different polymerases. As an example, when you use Phusion Polymerase, you have different way to calculate Tm, than when it is Taq Polymerase. Some Polymerase like Phusion have 2 buffers, like HF and GC, that will also ale a difference. So, check the polymerase, buffer and the Tm.

5) PCR conditions, again annealing temperature and extension depending on polymerase and primers.

There are many other small parameters that you can check, but if you make sure that everything mentioned above has been worked out for your gene of interest and amplification, then your PCR will definitely work.

Hope it helps. Best,

Simran

Hi, try putting a gradient PCR.. say from 55-65 degrees. The purity of primers and dNTPs  play a very important role. So make sure you buy it from a good company.

Try to add a few more cycles or to change annealing temperature.

Different primers often require different conditions. 

I would try in this scenario with increased cycle numbers and gradient temperatures between 52-62 and extended elongation time. 

Do check with the primer concentration and dntp concentration as well, some aliquotes may be bit more diluted. 

Hi Sailesh

Maybe you can use new aliquots of your reagents, sometimes the freezing-thawing cycles promote hydrolysis of dNTPS and salt preci[itation in buffers. Another point that could be important is the elongation time, if your amplification is less than 500bp try to use 15-20 sec. Extended elongation times produce primer extension of single strands depleting the available dNTPs in your reaction.

As template If you are using genomic DNA resuspended in TE or any other buffer, it can change the salt concentration when you put it in the PCR reaction, Yoy can elute with water after purification.

Best.

Pablo

 The same condition and reagents good for another gene just shows there was no problem with reagents. For PTGES genes, obviously, you should try different conditions, such as trying different annealing temperatures and MdCl2 concentration, etc.

hi 

Sailesh

Did you try to increase the amount of DNA and put gradiant ?

Hi,

I advice you to work on the annealing temperature with gradual temperature as the primers differ maybe you have a high GC. Also check the DNA concentartion, maybe you need to put more DNA to your sample. I also advice you to use Hotstar PCR set, it work perfect. Maybe you can do a little more thing by changing your ethedium bromide bath with a fresh one and give it more time (this is if you make ethidum bromide bath) otherwise use gel red it works perfect and safe. Good luck

Hi Sailesh

You have to optimize your primer's annealing temperature whenever you use a different set of primers. I suggest you to perform a gradient PCR to determine your primer's annealing temperature. However before performing  a gradient PCR, I try my primers to work 5 degree below than their Tm and they usually work at that temperature ( your forward and reverse primer's Tm differences should be ≤ 5) . After determining your primer's annealing temperature, you can utilize the same protocol (same dNTP, MgCl2 concentration) which worked correctly before. Good luck.

Basak

Dear Basak,

Thank you for your suggestion.

I also performed gradient PCR to optimize the annealing temperature. I got specific band but with very low intensity. Generally, when I do gradient PCR, I used to get high intensity band though not specific. But in this case, it is specific but low intensity.

  Dear Sailesh

If you don't get any primer dimers and the concentration of dNTP and MgCl2 gave the normal results in previous reactions before, I think the solution will be easier then. If I were you, before altering all the parameters, I would determine the temperature which gives slightly brighter band and I would perform a standard PCR with this Tm but with increased cycle number. Increased elongation time would also help, but it may cause unspesific bindings. Also you my decrease gel concentration a little bit, for example from %2 to %1.5. Hope it helps.

  Best

Different genes have different optimal PCR conditions. If it's band intensity, I would recommend checking the temperature and Mg concentration.

Try to do a gradient for temperature, could be the temperature is too high and limits annealing.

As for Mg, probably the Mg concentration is too low for optimum amplification of this gene.

Is your product GC rich

If yes than please try 3-5% DMSO with temperature gradient.

If no than use temp. gradient only