We have a BioRad T100 Thermal Cycler in our lab. I'm trying to set up a gradient PCR but the machine won't allow me to choose the temperatures in the gradient beyond the front and back row (low and high ends of range). I have 7 different PCR reactions that all have Tm's between 54-60C, and I'm wanting to set up a gradient with the front row at 54C, next row at 55C, and so on up to 60C at row 7, so that I can run all 7 samples (that have different Tm's) in one block in one night. However, the machine won't allow me to select the temperatures actually within the gradient - only the first and last temperatures - and I get undesired Tm's when I set the first row at 54C and the last one at 60C, 61C, 62C, etc. Is it possible to manually determine all the temps within the gradient? I'm not testing for a specific Tm, I already know the Tm's I need to use for these reactions (the PCR products will eventually be used in ligation reactions w/ T4 ligase). Is the gradient step designed this way on purpose? Or is there a way to get the temps I want? Anything helps, thank you!
Hello,
I have used T100 nad analogically to another thermocycles you can set there only the range of the gradient - temperature in first and the last row. If you want to obtain certain annealing temperatures for certain samples you need to try different sets/combinations of temperatures in first and last row during the programming procedure and observe the temperature values in the rows between them to choose the most suitable for your purposes (the exact values will be displayed on the screen of T100 as it was shown in post by Bin Mr).
Best wishes
Piotr
Bin Mr Piotr Androsiuk thank you for your input! I actually did try that method, of changing front/back temps to try and get the Tm's I wanted, but I didn't know if there was something I was missing as far as programming or settings. I'll try messing around with the beginning and end of the Tm range to get close to what I want - thank you both!
The gradient curve on the T100 is not linear, therefore it is almost impossible to obtain specific temperature in all different lanes. Check out the attached gradient profiles for a few different thermal cycler models (data from Thermo fisher). There are a few models in the market do better for this purpose such as the Biometra (Analytik Jena), which allows the user to set the intervals of the gradient and Veriti (Applied Biosystems), which has six individual temperature zones on the block.
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