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I am amplifying a gene where in a gradient pcr i am getting amplification at an annealing temperature about 5 degrees (67) higher than Tm (62.5)? What is wrong here? Also, I am getting a very strong intense band at wrong size for all the temperatures tried below Tm. Pls help

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    $\begingroup$ Are you calculating Tm bioinformatically, because that is just an estimate? If you have a lot of GC content, your melt temp will be higher. It could also be the salt concentration of your buffer. If they are old reagents, you might of had some evaporation and your salt content could be higher than normal... You also do not mention whether the wrong size is smaller or larger. Could be primer dimers or you could have off target annealing. primers do not need to be completely complementary to anneal properly and if you have a primer and a sequence with a lot of GC it could anneal preferentially. $\endgroup$ – AMR Nov 19 '15 at 13:13
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    $\begingroup$ Possible duplicate of Does an annealing temp higher than primer's Tm contribute to primer dimer? $\endgroup$ – AliceD Nov 19 '15 at 13:15
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It is very common to get annealing at higher than predicted Tm. If it's a problem in your case, especially if you're getting mis-annealing at lower temperatures, consider using touchdown PCR to find an optimal temperature. Or, pragmatically, if you have any flexibility in primer design, simply order a set of primers and try them all. Primers are cheap today, and you'll often find a set that work even where the design programs claim they won't.

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Tm of primers would not tell much about PCR conditions. When you get non-specific bands, it is worth to try higher temperature.

Your temp trying is already close to the temp to extend, so that shuttle PCR is available. Shuttle PCR has just 2 steps: 94 to 96 degrees for melting DNA and 68 to 74 degrees for annealing and extension together.

You might want to add DMSO or betain. They could help to reduce mis-annealing. If the non-specific bands are smaller and longer than bands you expect, longer and shorter extension times may give you better results, respectively.

And remember, primer design is also important. Check you have sequences repeating in your template and avoid to design primers from such sequences.


Regarding higher annealing temp than Tm, I often use T7 primer of which Tm is 48.3 degrees. The primer works well at 55 degrees of annealing temp. Usually, primers are excess in PCR reaction tubes, pushing the chemical equilibrium toward annealing. That is what I speculate.

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The TM of the primer is also influnced of the salt concentration of your mix. Do you try the PCR gradient? Or you try the calculation of Tm and take in consideration the salt concentration. http://www.biophp.org/minitools/melting_temperature/demo.php

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  • $\begingroup$ Welcome to SE Biology. It is a good idea to look at the date of posts — easy to miss, especially on a phone — because if you are asking a question of the poster or making practical suggestions in your answer you may be wasting your time. Of course if the question is scientific and you feel it has not been properly answered go ahead. (But not in this case, I think.) $\endgroup$ – David May 19 at 16:49

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