In short you would not expect to derive any meaningful PCR data from primers with Tms so far apart and for one primer with a Tm so low. Good primer design is crucial to successful PCR and the Tm is one aspect of that appraisal. In essence both of your primers should have Tms between 55C and 65C and should their tm difference should be no more than 2C. The annealing temp in the actual PCR is anything between Tm-2C to Tm-5C but Tm-2C will increase the potential specificity of your reaction. To have Tms of 55C-65C (even your primer @46.7C is arguably too low) your primer will probably be soemwhere between 18bp and 21bp in lenght with a GC content of 40%-60%. In addition you need to consider whether the primers will self anneal or bind to each other, i.e. potentially form primer dimers. There is alot on the internet about good primer design and alot of programs that will calculate your primer Tms and PCR reaction conditions. Find a link to a drop box of mine which provides a word tutorial prepared by me discussing optimal primer design and providing links in this word documant to programs which calculate primer TMs and other (annealing) matters mentioned

There is alos much on the internet once you have designed your primers as to how you set up optimal PCR reactions

I hope the above and my word document at least gets you started

https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0

I would say you cannot get any pcr products or many unspecific bands because of very low tm value

Increase primer length to increase Tm to about 58-60C

Or use a primer design package (available for free at several websites)

In short you would not expect to derive any meaningful PCR data from primers with Tms so far apart and for one primer with a Tm so low. Good primer design is crucial to successful PCR and the Tm is one aspect of that appraisal. In essence both of your primers should have Tms between 55C and 65C and should their tm difference should be no more than 2C. The annealing temp in the actual PCR is anything between Tm-2C to Tm-5C but Tm-2C will increase the potential specificity of your reaction. To have Tms of 55C-65C (even your primer @46.7C is arguably too low) your primer will probably be soemwhere between 18bp and 21bp in lenght with a GC content of 40%-60%. In addition you need to consider whether the primers will self anneal or bind to each other, i.e. potentially form primer dimers. There is alot on the internet about good primer design and alot of programs that will calculate your primer Tms and PCR reaction conditions. Find a link to a drop box of mine which provides a word tutorial prepared by me discussing optimal primer design and providing links in this word documant to programs which calculate primer TMs and other (annealing) matters mentioned

There is alos much on the internet once you have designed your primers as to how you set up optimal PCR reactions

I hope the above and my word document at least gets you started

https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0

There is big variation in your Tm 29.2 and 46.7.  You havenot design the primer properly, hence you will not find any amplification.  Go to Primer 3 software and there you will find option of Tm, so select the range of your forward and reverse primer between 50 to 55 or 55 to 60 or any value which suits your primer, but ensure that the Tm should be high, which would prevent hairpin formation in your primer.  After designing the primer either keep gradient PCR or you can reduce or increase 5 degree temperature either from your forward or reverse primer.  I am pretty much sure that you will get the amplification.

I agree with Lawrence and others. Your Tm values are too low to have specificity, as well as wide apart to have a meaningful data. A redesigning for primers keeping the Tm, GC content and primer length in mind is recommended. you may further find this attachment helpful. many sites like primer3  help you in designing primers. Many other like tmcalculator.neb.com, and other Tm calculator sites help you calculate theoretically feasible annealing temperatures to begin with.

To add to what I said above and expounded in my document

Try and keep primer Tms within 2C of one another and initially elect to use a Ta of Tm -2C in your PCRs. As already mentioned primer 3 is an excellent program for designing Tm compatible primers. I tend to take such primers and simply verify lack of self or primer diner annealing with programs mentioned in my word file on drop box. Alternatively and better still if your target sequence is a transcript design primers directly to genbank sequence using NCBI primer BLAST an absolutely superb program

finally for successful specific PCR try Tm -2C for your annealing temp in a standard 3 step PCR or better still if you design primers with matching TMs of 65C +/- 2C  try a 2 step PCR in which you have an initial denaturation cycle of 96C for 20 sec and a second cycle of Tm - 2 (as long at that Ta is above 60C) for 20s to 1 minute (upto 1kb) in which annealing and extension are combined into one cycle

if that doesn't work drop the Ta down to Tm - 5C in the context of above or as mentioned try a Ta gradient. Alternatively and my final word if standard PCR doesn't work try touch down PCR details of which can be found on google or a could supply if you sent me your chosen primer sequences 

First always run gradient PCR (30 - 47 C) to find out single temperature that suits the Tm for both primers. Then you can try  touchdown PCR program from 50 to 35 C with ramping of 1-2C per cycle.

I agree with the last speaker except with regard to the actual temperature gradient. Optimal PCRs are produced from primers with Tm of 55C to 65C so design primers as suggested by me and others such that your Tm resides in this region and then perform a gradient PCR within that span: If you are having to take the temp down to 30C that is too low for optimal PCR !!

Yoke Kqueen Cheah Laurence Stuart Dawkins-Hall Qurshid Hasan Khan Rasmus Goll Can any one tell me which temperature is perfect for getting band during PCR ? because I already test some temperature. Still I did not get band

• There is not such thing as a perfect Temp
• It is determined by experiment but generally Tm-2C is considered a good place to start
• If that doesn't work then the issue might not be temp; rather the quality and quantity of your starting DNA and/or primer design

I believe a melting temperature difference of 5 deg C between forward and reverse primers is apt for easy determination of the annealing temperature. Your Tm difference is too large and will affect primer annealing as explained by others. Please perform the screening test first and set out with the correct Tms that will favor successful PCR

Not sure abouy rationale for last answer

Tm-2c should work if your forward and reverse primer differ by upto 2c; and thats all they should differ by

That being so a temp gradient to determine optimum should be Tm-5c of Both primers (if compatible as described up to Tm+2c in 1c annealing temp increase; in other words 7 separate PCR reactions

ideal pair of primers should not have more than 5 degree C difference

What ? Primers should be NO MORE than 2C different if you describe them as ideal

Yes primers with a Tm mismatch of 5C can work but you greatly increase your chances of the PCR not workng and/or incuring secondary non specific products with this sort of mismatch

Gradient PCR is the best way to determine the annealing temperature.

change the primers