Hello i am trying a pair of new primers. Unfortunately I do not have enough reactions to perform a temperature gradient.
My HK primer has Tm of 60.3 (forward) and 60.8 (reverse) and the other primer I will use has 60.4 (forward) and 58.9 (reverse).
However it is also stated in the data sheet that my primer with 60.4 Tm has a strong secondary structure formation. I have seen that even though standard PCR kits have some amount of DMSO it still can be useful to add DMSO in order to reduce the Tm.
If so would you recommend additional DMSO and is 5% is a good starting point?
Also in this case can I blindly predict my Ta by subtracting 5 degrees from the lowest Tm I have?
Any help would very much be appreciated. :)
Best Regards
Hi
Before adapting anything I would give it a try with 60°C annealing temp. I guess it will work
Good luck
Sven
Generally, you should begin with an annealing temperature 3–5°C lower than the lowest Tm of the primers. Annealing temperature may need further optimization, depending on the amplification results. For instance, if there is no or low amplification, the annealing temperature may be lowered in increments of 1–3°C during optimization. If nonspecific PCR products appear, however, the annealing temperature can be raised in increments of 1–3°C (up to the extension temperature) to enhance specificity.
The presence of DMSO lowers the Tm of the primer-template complex, e.g. 10% DMSO can decrease the annealing temperature by 5–6°C. In this case, the annealing temperature should be adjusted accordingly. During initial stage, I will suggest not to add additional DMSO. Just start with annealing temperature 5°C lower than the lowest Tm of the primers.
Try a gradient of annealing temperatures to figure out which one is the best. Don't start adding other components like DMSO until you've tried a gradient.
Thank you all for your answers and interest.
I will consider your points during my experiments.
Best Regards :)
Hi,
as Bhairavi Vajaria suggested, you should start few degrees below the Tm.
However, in qPCR usually you try to use the same annealing Temp (60ºC) for all your primer pairs, so that you can analyse your samples in the same plate for different targets.
In your specific case you can try 60ºC, you will probably get the correct amplification (check the melting curve). Then, I suggest you calculate the Efficiency of your primer pair by performing amplification of any sample after preparing a serial dilution of it (2-fold will do, at least 4 different points). you will find instructions for calculating the efficiency online.
Efficiency should be ideally 100%, but still acceptable from 90 to 110%. This ensures that at each cycle you have doubling of your target DNA.
Next time you design a primer pair, try to have both of them with a Tm of 60ºC, by adding one base, possibly A or T (if there is in the sequence surrounding the oligo you are selecting) to the one with the lowest Tm. Good luck!
You can try to run a gradient PCR using the cDNA and the primers. Then use the temperature that would give you the best result.
Hi, I fully agree with the recommendations above. Just to add more tips: In my experience, PCR (not qPCR) usually works well with much higher annealing temperatures than the predicted Tm. Also, different DNA oligo synthesis providers use different algorithms to predict the Tm, some of them very obsolete. The predictions may vary by more than 5 degrees or even much more if salt and magnesium concentrations are not reflected. To confront your datasheet values with the most accurate Tm prediction algorithms, check the Primer Inspector online tool:
http://www.molbiotools.com/primerinspector.php
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