Hi Md,

There are a few troubleshooting tips you can try. Consider another primer pair (positive control) known to amplify with your template. If this does not work you may have fragmented gDNA or PCR inhibitors in the gDNA or other contaminants in the polymerase reaction components. If the positive control works, try a gradient PCR with various annealing temperatures, a touchdown PCR and/or additional MgCl2 may also help. All the best

Just to add a little something to Joshua Philips ' excellent answer. I haven't used Q5 but in my experience all those high-fidelity proofreading polymerases are finicky regarding Mg concentration. I would first set 4 or 5 reactions at different Mg2+ concentrations before trying more complicated solutions...

Hope it helps!

In addition to suggestions given by Joshua Philips you can try using annealing temperature for primers 3-5 degrees less than the average Tm of both primers. Try not to go much below this range to capture the desirable size product. Gradient PCR should work out for you (of course use a +ve and -ve control)

Thank you all. I have used gradient PCR and found the expected PCR product.

Md Atikur Rahman

Good to know you got the band. So, what went wrong at the beginning? The Tm you set?

Yuan-Yeu Yau I got the expected result in different Tm from the recommended value (Tm from NEB Tm calculator). It was less than the recommended Tm.

There is always confusion about what Tm means for some reason and I would have expected what you have observed - that an annealing temperature of less than the calculated Tm works best. Its always been that way. I recommend you download snapgene viewer (free) and read what it says about Tm values:

"The SnapGene Tm calculator assumes that the buffer has 50 mM Na+ and no Mg2+. With this calculation method, when PCR is performed using a traditional polymerase such as Taq, the optimal annealing temperature for the PCR reaction is about 0–5°C below the primer Tm values.

For some newer PCR polymerases, including Phusion®, Phire®, Q5®, and PlatinumTM SuperFiTM, the optimal annealing temperature is higher than the primer Tm values. The reason is that these polymerases are fused to a processivity–enhancing dsDNA-binding domain that stabilizes the primer-template complex.

For such polymerases, the approach recommended by New England BioLabs and Thermo Fisher is to use older thermodynamic parameters to calculate the Tm values. Those older parameters are less accurate, and they give Tm values higher than the ones obtained with the up-to-date parameters.

The SnapGene team has decided not to provide inaccurate Tm values. Instead, we recommend that when a polymerase with a dsDNA-binding domain is used, the annealing temperature for the PCR reaction should be about 6–12°C (for Phusion or Phire or Q5 polymerase) or 4-8°C (for Platinum SuperFi polymerase) above the primer Tm values calculated by our software.

Printed from SnapGene®: 13 May 2021 09:38

SnapGene calculates oligo melting temperature (Tm) values using a nearest neighbor thermodynamic algorithm with up-to-date parameters [1]. This method is the most accurate one available [1,2]. For a given duplex, the calculation accounts for any internal mismatches, loops, and dangling ends.

For a primer binding site, nearest neighbor calculations are combined with dynamic programming [3] to find the most energetically favorable duplex. Because detailed duplex structures can be distracting, SnapGene shows simplified duplexes by default, but you can see full duplex structures with a setting in Preferences.

Assumptions

Limitations

References

Printed from SnapGene®: 13 May 2021 09:38"

Glad to hear the lower Tm worked for you. Just as a comment - DNA concentration is another thing you might want to consider in further projects. You wrote that you use 100 ng/µl template. Do you mean 100 ng DNA per µl pcr reaction volume? That seems excessive. It has been shown that excessive amounts of DNA may inhibit amplification. Thus, you could avoid possible problems by lowering the DNA concentration. I usually use 0.5-1 ng/µl DNA to amplify single genes in large plant genomes.

Best wishes,

Klaus