primers TM - (Jun/24/2007 )
hi
on optimizing PCR products i use annealing Temp thats above or below the TM of the primer , but one of my collegues told me that i shouldnt be using annealing highr than the TM !!!!
is that true ? and why ?
Your annealing temperature should be below the tm.
the Tm is the temperature at which half your primers are annealed to their complementary strand (template). Above the tm, and you get less then half. And the fewer molecules you have annealed to the template, the lower you PCR yields will be.
It is true however that sometimes you get even better product with a annealing higher than primers Tm for some reasons.
And my colleague told me, that some assays even require higher temperatures to be working optimaly. But that is not a general guideline.
I usualy try classic 4 degs below Tm (as counted by primer3) as a first hit and then optimize further.
Set annealing in a range from lower temperatures to those close to Tm and if you see decline in the amount or quality of product, you simply choose the best temperature. But if you set this and product seems to be beter and better, then continue with higher temperature as you wish.
But generaly try only those under Tm.
My understanding was that it depends on the enzyme and the primer size. Also, there are several different ways to calculate the Tm. I go to the website of the primer I am using and calculate the primer Tm by their method, then use the product insert to determine the annealing temperature I will use.
Actually, that is a bit of an exaggeration- i actually just use my standard PCR cycle with an annealing temp of 53C, and if that doesn't work, then I start fiddling around with the annealing temp 
Hi, the easiest way to find the optimal Annealing temperature is to do a gradient PCR. Sometimes , the optimal Annealing temperature can be higher than Tm. For example, the Tm of one pair of my primers was about 55C. But the optimal Annealing temperature was acutally 58.8C , as shown by the gradient PCR. So , I chose 58.8 as the annealing temperature and got very good results.
on optimizing PCR products i use annealing Temp thats above or below the TM of the primer , but one of my collegues told me that i shouldnt be using annealing highr than the TM !!!!
is that true ? and why ?
When temperatures under Tm are not working properly I also do a gradient PCR , I mean...I set the PCR to increase anealing temperature in each cycle.....And most of the times it solve the problem....
But I also have a question...when you do this, how do you know which is the optimal TM.?...because the machine increase the temperature each time...It is not easy to find out about it? 
But I also have a question...when you do this, how do you know which is the optimal TM.?...because the machine increase the temperature each time...It is not easy to find out about it?
I think what you are referring to is a touchdown PCR, which is different to a gradient PCR. With a gradient PCR you set each column of wells/positions in your PCR machine to a defined temperature, then when you run products on a gel to determine which one worked, you will have recorded what position in the machine that tube was- and that is your optimal temperature.
Hope that makes sense
could u please tell me how to do gradient PCR ?
and what type of pCR device can do it?
I use a PCR cycle programed on our Biorad icycler that was created by another member of the lab so I don't actually know how to set one up from scratch. You could try consulting your machine's manual, or speak to a rep from the company who made it??