I have seen some protocols about q-pcr.
I find that the primer and sample are same,but the annealing temperature of q-pcr and standard pcr are not same.
And the q-pcr is higher than standard pcr.
why do the q-pcr have a higher annealing temperature?
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6 replies to this topic
#2
Trof
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Posted 10 August 2011 - 08:53 AM
There is one widely used qPCR annealing temperature and that is 60 °C. Usually you design primers and then find optimal annealing temperature, in real-time PCR you design primers to work at 60. I'm not really sure what is the reason, but as this is higher that the classical starting annealing temperature (55 °C) it may be because the increased need for specificity. Higher annealing is more specific and DNA stays better denatured. I design all my "classic" PCR primers for 60 now, and I really love the results, clean and efficient.
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#3
Adrian K
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Posted 11 August 2011 - 10:14 AM
Trof ,is it still acceptable if the temperature up to 63C ??
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"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#4
hobglobin
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Posted 11 August 2011 - 11:10 AM
you can be even more higher and then make a two-step PCR with a step of simultaneous annealing and extension. The temps should be around 68°C then, if I remember right (never tried it out so far myself).
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#5
Adrian K
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Posted 11 August 2011 - 09:48 PM
Yeah, Brilliant Dr H!
I should have think of that... my gradient have yet to reach the temperature... ok I will try later in the lab.
Cheers!
Adrian
I should have think of that... my gradient have yet to reach the temperature... ok I will try later in the lab.
Cheers!
Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#6
Trof
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Posted 11 August 2011 - 10:37 PM
Adrian K, on 11 August 2011 - 10:14 AM, said:
Trof ,is it still acceptable if the temperature up to 63C ??
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
I never trust anything that can't be doubted.
#7
Adrian K
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Posted 12 August 2011 - 11:18 AM
Hi All, I just try my new assay today... I try up to 66C.... eliminated the unspecific binding seen by naked eyes.
But I still yet to get rid of primer dimmer problem... I don't have probes. the assay is mean for Evagreen.
But I still yet to get rid of primer dimmer problem... I don't have probes. the assay is mean for Evagreen.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
