c******e
发帖数: 350 | 1 Sorry, but I don't have Chinese input here.
We have an old thermocycler (for PCR in capalliry) recommend a ramping rate
as high as 20C/sec.
When we ran the same PCR using a 96-well plate on regular thermocycler with
the regular ramping rate (2C/sec?), amplification was failed.
I wonder whether the failure is due to change in ramping rate? How does
ramping rate affect PCR?
Thanks a lot! | T****O
发帖数: 407 | 2 My guess is there is a upper and lower limit that the machine could reliably
control the temperature temporal and spacial gradients. If your ramping
rate is so high that there'd be regular heat spikes, you kill your enzyme (
probably everything) and break the reaction. If it's too low, then the
temperatures required by reaction could not be achieved or maintained and
your reaction fails as well.
rate
with
【在 c******e 的大作中提到】
: Sorry, but I don't have Chinese input here.
: We have an old thermocycler (for PCR in capalliry) recommend a ramping rate
: as high as 20C/sec.
: When we ran the same PCR using a 96-well plate on regular thermocycler with
: the regular ramping rate (2C/sec?), amplification was failed.
: I wonder whether the failure is due to change in ramping rate? How does
: ramping rate affect PCR?
: Thanks a lot!
| l**********1
发帖数: 5204 | 3 HOT-START PCR is their key point.
from primers or template some misannealing during the initial heating step.
//www.roche-applied-science.com/PROD_INF/BIOCHEMI/No1_00/PDF/page35-36.pdf
rate
with
【在 c******e 的大作中提到】
: Sorry, but I don't have Chinese input here.
: We have an old thermocycler (for PCR in capalliry) recommend a ramping rate
: as high as 20C/sec.
: When we ran the same PCR using a 96-well plate on regular thermocycler with
: the regular ramping rate (2C/sec?), amplification was failed.
: I wonder whether the failure is due to change in ramping rate? How does
: ramping rate affect PCR?
: Thanks a lot!
| c******e
发帖数: 350 | 4 Thanks.
But maybe I didn't explain well what happened.
We set up 2 reactions with identical reagents on 2 thermocyclers as below.
thermocycler Capillary 96-wells
Ramp rate (C/sec) 20 2
PCR volume 10 ul 10 ul
Amplification success fail
The reason we changed the ramp rate is that 2C/sec is recommended for the
96-well thermocylcer.
We didn't change reagents or Taq, because they are our successful standard reagents for any PCR including the ones on the 96-well thermocycler.
So we thought that the reaction failure may due to the change in ramp rate?
Or would it be the small volume (10ul)? We usually use 20ul in the 96-well?
Thanks
reliably
【在 T****O 的大作中提到】
: My guess is there is a upper and lower limit that the machine could reliably
: control the temperature temporal and spacial gradients. If your ramping
: rate is so high that there'd be regular heat spikes, you kill your enzyme (
: probably everything) and break the reaction. If it's too low, then the
: temperatures required by reaction could not be achieved or maintained and
: your reaction fails as well.
:
: rate
: with
| c******e
发帖数: 350 | 5 Do you suggest that with the 20C/sec ramp rate, the reaction is like a hot
start, so when we changed the thermocylcer and ramp rate to 2.2C/sec, the
reaction failed without hot start?
I would like to try hot start on the 96-well thermocycler.
Thanks,
【在 l**********1 的大作中提到】
: HOT-START PCR is their key point.
: from primers or template some misannealing during the initial heating step.
: //www.roche-applied-science.com/PROD_INF/BIOCHEMI/No1_00/PDF/page35-36.pdf
:
: rate
: with
| n********k
发帖数: 2818 | 6 If one uses takara PS enzyme, switch from a ramp rate 20c/ sec to 2.2c/sec,
using a same protocol the chance for a failure is very high...if your
protocol was not robust, any change could just ruin it...It depends on so
many different factors... the ramp rate could matter a lot or nothing...only
yourself would know/figure why...the key is to know your system and know-
how...don't always wait to know a tech in depth until one runs into troubles
...many of those painful/costly failures could be avoided altogether with a
little more effort on reading etc...
【在 c******e 的大作中提到】
: Do you suggest that with the 20C/sec ramp rate, the reaction is like a hot
: start, so when we changed the thermocylcer and ramp rate to 2.2C/sec, the
: reaction failed without hot start?
: I would like to try hot start on the 96-well thermocycler.
: Thanks,
| l**********1
发帖数: 5204 | 7 又一个 搞Protocol创新的
微细管PCR protocol 的 10 ul 用ABI 9700/9600 PCR thermocylcer 干
试出没? by HOT START
If positive next submit to
Nature Methods ? just guess
【在 c******e 的大作中提到】
: Do you suggest that with the 20C/sec ramp rate, the reaction is like a hot
: start, so when we changed the thermocylcer and ramp rate to 2.2C/sec, the
: reaction failed without hot start?
: I would like to try hot start on the 96-well thermocycler.
: Thanks,
|
|
