Hi,
I got a PCR program that has as final extension step (at least I interpret it as extension step) 30 minutes at 60°C. Normally I use 10 minutes at 72°C...
So what is the aim of this long time at 60 degrees?
(Complete program:
3 min 94°C
30 s 94°C
90s 58°C
60s 72°C (loop back to step 2 30 times)
30 min 60°C
Thanks for any ideas...
Submit your paper to J Biol Methods today!
2 replies to this topic
#1
hobglobin
-
- Global Moderators
-
- 5,446 posts
Growing old is mandatory, growing up is optional...
84
Excellent
Excellent
Posted 17 August 2009 - 03:18 AM
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
#2
noelmathur
-
- Active Members
-
- 72 posts
Enthusiast
0
Neutral
Neutral
Posted 17 August 2009 - 06:07 AM
Frankly, quiet odd to my knowledge. I always do as you say.
But why do you need annealing time to be for 90sec?, 30 sec is enough, right?
Would love to hear what others have to say.
But why do you need annealing time to be for 90sec?, 30 sec is enough, right?
Would love to hear what others have to say.
#3
swanny
-
- Active Members
-
- 367 posts
Veteran
8
Neutral
Neutral
Posted 17 August 2009 - 05:16 PM
AT 60 degrees, most of the thermophilic polymerases would only work very slowly. Perhaps it's there for some kind of proofreading (wouldn't work with Taq, though), or to ensure all amplicons are full length (but your point of 10 minutes @72 deg would do the same thing).
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.
