For the amplification of my target gene, I ran a Gradiant PCR by keeping all other components of the PCR mixture constant, and only altering the Tm. I set the Tm values at 52°, 53°, 54° and 55°. To my astonishment, I got my band for each of these Tm values. Isn't the Tm specific for every gene? What do these results depict? Do all of these bands contain the amplified sequence of my gene or is this any case of non-specific primer binding? I would be highly grateful for the response.
Safeena Aslam it is clear that your primer anneal at all those Tm values you set up your gradient with. This is not surprising at all, given that your set temperatures are on the lower end of the spectrum.
the higher annealing temperature you set, the more specific binding your reaction will favor. So try 55 (as you know it works), 57, 59, 61 and 63C next time. Likely that you will find the most specific temperature somewhere between there.
good luck!
Tm is important ragarding primer anneling and is less relevant in gene specificity. A primer set can have a broad range Tm. You have to find out at which temperature the band is brightest (more amplified).
I agree with the above that generally if a primer set works at any temperature it will work at all temperatures below that temperature but may start to amplify more bands as the temperature drops
Looking at your picture you have a strong primer dimer which will make your amplification less efficient and may effect sequencing if that is your next step so you may want to use less primer or a hot start polymerase. The dimer may look faint but it is short so traps less ethidium bromide so a lot of material still looks faint
@Paul Rutland Yes Sir my next step is sequencing of this gene. Surely I will try both of these recommendations by you to get a more amplified sequence of my gene. Thanks a lot for the kind response.
As others have said, it just appears that your primers have a broad useful annealing range. These are most likely all the exact same PCR product, but you could always sequence them if you have a reason to think that you have more than one product (closely-related genes, expanded gene family, etc.). Honestly, the most reliable primers are typically ones with a wide range of annealing temps.
Safeena Aslam
The gene has a rate of temperature and may vary depending on the type of substances or conditions .....
All the comments above plus the room temprrture during PCR runing and precised steps
Regards
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