The PCR protocol says after 15min at 95C, there was a 5% ramp down to 57C. I'm familiar with a touchdown PCR, but this terminology is new to me. Does this mean the annealing step starts at 95C and decreases to 57C? Or is there missing information for the range of the annealing temperature?
The PCR machine I'm using (Eppendorf Mastercycler) does not have an option for a percent ramp (only C/sec)- how would I change this to ramp rate if I need to?
Ramping is the rate at which a thermocycler can heat and cool a block. You can think of it as the slope of a plot of temperature as a function of time. A 5% ramp means that the machine is heating/cooling at 5% of the average maximal rate. So what they are trying to do is denature everything in the tube, then slowly cool the reaction down to the primer annealing temperature to favor all thermodynamic complexes rather than kinetic, misannealed complexes. This is probably important since you must be having primer specificity issues to be trying this method.
Depending on the cycler, there will be different means to adjust the ramp rate. On an Eppendorf Mastercycler, hover over the temperature, then hit options, this will bring up a submenu where you can designate the ramp rate. Make sure to select the correct temperature that you are ramping to.
Thermo has a nice description of cycling terminology that I have attached.
As a side comment, the 15 min denature at 95C seems a little excessive. Be careful about long denaturation times at high temperatures as the triphosphate stability is not so great at 95C and it could drastically effect your PCR efficiency.
https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/PCR/pdfs/Thermal-Cycler-Ramp-Rates-AppNote.pdf
Ramping is the rate at which a thermocycler can heat and cool a block. You can think of it as the slope of a plot of temperature as a function of time. A 5% ramp means that the machine is heating/cooling at 5% of the average maximal rate. So what they are trying to do is denature everything in the tube, then slowly cool the reaction down to the primer annealing temperature to favor all thermodynamic complexes rather than kinetic, misannealed complexes. This is probably important since you must be having primer specificity issues to be trying this method.
Depending on the cycler, there will be different means to adjust the ramp rate. On an Eppendorf Mastercycler, hover over the temperature, then hit options, this will bring up a submenu where you can designate the ramp rate. Make sure to select the correct temperature that you are ramping to.
Thermo has a nice description of cycling terminology that I have attached.
As a side comment, the 15 min denature at 95C seems a little excessive. Be careful about long denaturation times at high temperatures as the triphosphate stability is not so great at 95C and it could drastically effect your PCR efficiency.
https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/PCR/pdfs/Thermal-Cycler-Ramp-Rates-AppNote.pdf
If your thermocycler does not offer the option to do this automatically, you would need to decrease the temperature by about 2 degrees per minute. It's a 38 degree difference between 95 and 57 degrees, so this would take about 20 minutes for the annealing step. That seems really slow, but perhaps your primers lack specificity.
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