Difference in melting temperature - (Jun/04/2015 )
Hello,
I have a question concerning the melting temp. of the designed primers. I design my primers using Primer3plus. For example: melting temp. of a primer on Primer3plus shows to be 60 and when i check the melting temp. on Sigma (oligo evaluator) or basic.northwestern.edu primer melting temperature they are different (63.8 and 62.6 respectively). Why is there such a variance? And in this case which Tm should be trusted regardless of running a gradient.
Thanks
None of the above, you need to determine the best Tm (actually T annealing, not T melting) empirically.
bob1 on Thu Jun 4 10:39:22 2015 said:
None of the above, you need to determine the best Tm (actually T annealing, not T melting) empirically.
And how do we determine that?
Traditionally to determine best annealing temp you would do a gradient PCR.
However in my cloning work (making expression constructs), I always use touchdown PCR to amplify anything. It removes all the headaches associated with melting temps.
At this point I don't bother checking melting temps anymore or secondary structure prediction. I routinely use primers with 20C difference in melting temp with touchdown PCR no trouble.
Mad Researcher on Thu Jun 4 11:30:17 2015 said:
bob1 on Thu Jun 4 10:39:22 2015 said:
None of the above, you need to determine the best Tm (actually T annealing, not T melting) empirically.
And how do we determine that?
Experimentally, trial and error/gradient. The difference in the different programs may well be down to the assumptions that the different programs make about the salt in the reaction, the amount of which affects the annealing temperature. Usually you can take a Ta and use a Tm of about 5-10 degC lower.
bob1 on Thu Jun 4 22:54:33 2015 said:
Mad Researcher on Thu Jun 4 11:30:17 2015 said:
bob1 on Thu Jun 4 10:39:22 2015 said:
None of the above, you need to determine the best Tm (actually T annealing, not T melting) empirically.
And how do we determine that?
Experimentally, trial and error/gradient. The difference in the different programs may well be down to the assumptions that the different programs make about the salt in the reaction, the amount of which affects the annealing temperature. Usually you can take a Ta and use a Tm of about 5-10 degC lower.
Is there a possibilty to run a gradient on qPCR system (Stepone plus). Also, is it necessary to run an elongation step on qPCR?
I only run a 95°C 10 min, 95°C 30 sec, 60°C 30 sec for 40 cycles. I didn't get anything.
So, now i want to include 95°C 10 min, 95°C 30 sec, 60°C 30 sec and 72°C for 40 cycles (or should i make it 45 cycles)?