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How does the ramping rate affect PCR?

How does the ramping rate affect PCR?# Biology - 生物学

o*t2012-02-10 08:02

1 楼

又是一个冬季，阴阴的天，只是少了些雨
有些事情，刻意的去回避，或是想去忘记，却在某时某刻的突然想起
你的承诺。我们的故事，多像歌里的画
不知为何会想起用这首歌来征文，一别经年，许久未联系，在这个冬季，在这Love版，向所有人征文-----你的承诺...........
熟悉的歌，只是已不再熟悉的你和我。想起我们曾在一起的点滴，还有现在我们这最熟悉的陌生人，心里会失落，只是不再有伤与痛
有些事，放开了就好；有些人，释然了就不再恨
我想，未来的某天或许会再遇见。再遇见你，希望我会微笑的问你一句：好久不见，都还好么？
如烟往事，再回首，往事如梦
谨以此文，纪念某年的某个冬季，某年的某某和某某的故事
从此，你我不相干

n*s2012-02-10 08:02

2 楼

今天路过t-mobile店面，看到其现在推出一个$49.99 unlimited voice+text+data(
first 2gb at 4g speed) per line的family plan。应该是最新的plan，还挺有吸引力
的。
正在考虑是不是转成这个，有兴趣的可以去t-mobile瞅瞅

c*e2012-02-10 08:02

3 楼

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!

w*n2012-02-10 08:02

4 楼

某年某月某个承诺
如花如梦如烟往事
★ Sent from iPhone App: iReader Mitbbs Lite 7.28

v*s2012-02-10 08:02

5 楼

我也喜欢这个，可惜不是individual的

T*O2012-02-10 08:02

6 楼

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!

o*t2012-02-10 08:02

7 楼

呵呵某年某月

【在 w**********n 的大作中提到】

: 某年某月某个承诺
: 如花如梦如烟往事
: ★ Sent from iPhone App: iReader Mitbbs Lite 7.28

G*h2012-02-10 08:02

8 楼

看起来不错
偶是不是考虑加入之
比两个 prepaid phone 要省钱了

【在 v***s 的大作中提到】

: 我也喜欢这个，可惜不是individual的

l*12012-02-10 08:02

9 楼

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】

d*r2012-02-10 08:02

10 楼

再回首，云遮断归途，再回首，荆棘密布

e*t2012-02-10 08:02

11 楼

1. 49.99 per line;
2. no subsidized phones.

【在 n********s 的大作中提到】

: 今天路过t-mobile店面，看到其现在推出一个$49.99 unlimited voice+text+data(
: first 2gb at 4g speed) per line的family plan。应该是最新的plan，还挺有吸引力
: 的。
: 正在考虑是不是转成这个，有兴趣的可以去t-mobile瞅瞅

c*e2012-02-10 08:02

12 楼

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

M*e2012-02-10 08:02

13 楼

从此，你我不相干
这句话说起来号潇洒，做起来好心痛

G*h2012-02-10 08:02

14 楼

原来还是js

【在 e*****t 的大作中提到】

: 1. 49.99 per line;
: 2. no subsidized phones.

c*e2012-02-10 08:02

15 楼

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

w*n2012-02-10 08:02

16 楼

必须的就要这么“绝情＂
★ Sent from iPhone App: iReader Mitbbs Lite 7.28

v*s2012-02-10 08:02

17 楼

你看错了吧，两条线要100呢

【在 G*****h 的大作中提到】

: 看起来不错
: 偶是不是考虑加入之
: 比两个 prepaid phone 要省钱了

n*k2012-02-10 08:02

18 楼

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,

o*t2012-02-10 08:02

19 楼

释然了就好

【在 M**e 的大作中提到】

: 从此，你我不相干
: 这句话说起来号潇洒，做起来好心痛

n*s2012-02-10 08:02

20 楼

2 在哪儿看到的？这个还是要2-yr contract的啊

【在 e*****t 的大作中提到】

: 1. 49.99 per line;
: 2. no subsidized phones.

l*12012-02-10 08:02

21 楼

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】

e*t2012-02-10 08:02

22 楼

it's on tmobile's website and you can ask tmobile store too.

【在 n********s 的大作中提到】

: 2 在哪儿看到的？这个还是要2-yr contract的啊

s*n2012-02-10 08:02

23 楼

tmobile信号不好，不在乎的时候没什么，真的有急事打不出接不到电话会急死人。我
用tmobile的prepaid，前几天碰到几次，现在想换page plus了

r*t2012-02-10 08:02

24 楼

这个 per line 不可能省钱, 我看来看去这个都是最贵的那种 plan, 不是有 $30 一个
月的么，再往下还有 prepaid 更省。

【在 G*****h 的大作中提到】

: 看起来不错
: 偶是不是考虑加入之
: 比两个 prepaid phone 要省钱了

w*72012-02-10 08:02

25 楼

tmobile的信号的确一般。

y*f2012-02-10 08:02

26 楼

可以加。
subsidized phones 500分钟每月5块。 unlimited data每月只要10块。挺便宜的。

【在 e*****t 的大作中提到】

: it's on tmobile's website and you can ask tmobile store too.

y*f2012-02-10 08:02

27 楼

信号是差，靠便宜拉人。

【在 w*********7 的大作中提到】

: tmobile的信号的确一般。

N*m2012-02-10 08:02

28 楼

看地方，我这里还是最好的

【在 s****n 的大作中提到】

: tmobile信号不好，不在乎的时候没什么，真的有急事打不出接不到电话会急死人。我
: 用tmobile的prepaid，前几天碰到几次，现在想换page plus了