I have ordered primers from Thermo fisher scientific and in the analysis sheet it has been mentioned two tm
The first one is tm 1M na+ 70 and the second is Tm 50mM na+ 48 and each has different temperatures which of them should I use to adjust the anneling Temp for my PCR?
The second one will be your annealing temp of each primer.
Do you mean that at 48 my primer will work ? What is the maximum anneling temp limit? at this temperature I will definitely have high number of non specific bands even after using hot start master mix what should I do then ?
Tm mentioned in the analysis sheets always doesn't help in calculating Ta. I would suggest you to run a gradient pcr with temperatures ranging between 46-56. The Ta is lower than the Tm of the primer which has a lower Tm amongst the 2primers.
I see, but my point is that my primer pair have 4 tm not 2
Forward tm 1 M Na 71-tm 50mM Na 49
Reverse tm 1M Na 70- tm 50mM Na 48
Which should I use in this situation Tm 1M or Tm 50mM?
In the analysis sheet always write two Tm. one for forward primer and one for reverse primer. It doesn't mean you get results at that temp. Sometimes 3 degree on the lower or above side of average Tm of forward and reverse primer is the annealing temperature. You can perform a gradien pcr with rising annealing temperature as Tushar Chakraborty said. You can also use Tm calculator software on the Thermofisher website or other websites. You can try several online calculators and compare the answers you get.
Asmaa Nasr Just focus on Tm 50mM and run a gradient between 45-60 or use online software
Use Tm mentioned for 50mM. To be sure, run a gradient pcr as I suggested before.
I suggest you run at 44 in your case. If you run higher, you probably get no reaction. If you go lower (below 42), you will see non-target bands.
good luck Asmaa Nasr
Try the Tm calculator from Thermo Fisher.
https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
Calculate expected Tm of the primer at designing stage. Ta will be less than Tm by at least 3 degrees. Both depends on the number of C+G. There is a chance of nonspecific annealing if Ta is set at very low temperature compared to Tm.
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