Currently I have designed a few sets of primers, most of them the Tm is 69-79C. So is this okay since some of the Ta are higher than the temperature of polymerase.
I agree with Imtiyaz Khan, it is better to design your primers at Tm lower than 72.
However, often we need to amplify regions that are high GC, and we do not have a choice of moving primers to adjust the Tm. You can shorten the primer to lower the Tm down to 68, or you can also use the Deep VentR DNA Polymerase from NEB.
The calculated half-life of Deep VentR DNA Polymerase at 95°C is 23 hours. You should be fine using your primers with that DNA polymerase.
https://www.neb.com/products/m0258-deep-ventr-dna-polymerase
Its good if you are designing your primers with Tm 55' to 65' C.
Ta of more than 72'C is not suggested, nevertheless you can try with gradient temperature below polymerase activity for your primers.
maximum you can use 2 step PCR protocol for your primers etc.
Altenatively you can use DMSO to lower down Tm of your primers.
Some high fidelity polymerses can help you to amplify your gene.
https://www.neb.com/.../m0530-phusion-high-fidelity-dna-polymerase
add more information to get more info.
all the best
I agree with Imtiyaz Khan, it is better to design your primers at Tm lower than 72.
However, often we need to amplify regions that are high GC, and we do not have a choice of moving primers to adjust the Tm. You can shorten the primer to lower the Tm down to 68, or you can also use the Deep VentR DNA Polymerase from NEB.
The calculated half-life of Deep VentR DNA Polymerase at 95°C is 23 hours. You should be fine using your primers with that DNA polymerase.
https://www.neb.com/products/m0258-deep-ventr-dna-polymerase
I am doing inverse PCR to amplify my full length gene from metagenomic sample. The primers have to specific and long, so that's why the Tm are very high. Currently I am using Takara PCR kits. By the way, may I know more info on two-steps PCR?
A two-step PCR combines the primer annealing and elongation step, so that you have only denaturation and the combined annealing / elongation step.
On what basis is your Tm temp determined ? The only absolute method is to make a complementary primer, anneal and so a melt curve analysis with Sybr green in a real-time PCR machine in PCR buffer if you have access to one. You can then determine the Tm. The recommended annealing temp is usually 3-6C less. If you have a melt curve, you can then add increasing concentrations of DMSO / glycerol etc to see if you can bring it down by several degrees. Calculated Tms for long oligos are not particularly accurate. You may find that with very long primers that they actually behave like a mixture of primers with "low " and "higher" melting regions.
Ian Teo
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