s**u
发帖数: 9035 | 1 最近，我们实验室购买一台Bio-rad CFX96 real-time PCR， 用一个月后，数据不稳定
，特别是边缘效应明显，A12--H12数据偏高，中间也有数据乱跳的现象。公司派了一个
Engineer检查，说产品合格，Lid压力偏低，可能导致Plate压不紧，液体挥发。建议更
换新的Lid， 可是地区Manager不同意，说完全合格。
只几天8/27，8/28 作8块plate,效果还是比较好，8/29连续2块Plate均不好，数据乱跳
。我们现在每次Run完，均需要让PCR shut down15 分钟再继续Run,可是还是很不稳定
，正是愁死我了。我们有大量临床Sample。
请教使用过Bio-rad CFX96 realtime PCR的朋友们，仪器不稳定的原因有哪些？如何解
决？我们3人操作绝对没有问题，以前使用Biorad-IQ5也有同样问题。 |
A******y
发帖数: 2041 | 2 What kind of plate are you using? It looks like you didn't seal the film
properly. The best plate is white semi-skirt. If you need the PCR to shut
down 15 mins, are you sure you cool down the plate after the run i.e. around
4 degree. Also, did you prep your plate on a well cooler?
There are also one possible problem. Did you check the linearity of your
primer? It is extremely crucial and I know a lot of lab don't do it (which
is hilarious). Primary simple should jump around a bit, and the N is your
friend, unless your technical repeat is not repeatable. |
s**u
发帖数: 9035 | 3 We use Bio-rad plate and Bio-rad film as they recommended.
I sealed the film properly each time, but sometimes I found solution
evaporate at A12 to H12 (right side), I don't know why.
I prepared my plates on a well cooler and put on ice.
I got an email from Bio-rad Technical service below this morning(8/31/11 9;
22 am):
I just wanted to send you an update on the status of your CFX96 case.
As I conveyed to you yesterday during our phone call, your situation is one
that we have never observed and has reached the highest level of our
technical support chain. Discussions are ongoing regarding how we can come
to the best resolution for your instrument's performance, though no decision
has been made at this point. |
s**u
发帖数: 9035 | 4 We use Applied Biosystem MicroRNA commericial primer, the primer are all
good.
We just find solution evaporation on 2 plates on 8/29/11, one is FAM, one is
SYRB plate, and it never happened before although I run more than 100
plates using IQ5 and 30 plates using CFX-96.
还有这台CFX-96是作为公司demo用的，原价29，000， 因为作为demo用了半年，所以价
格为25，000，我们7月1日才刚刚购买拿到仪器。7月14日才刚刚完成培训。结果2-3个
星期就发现问题，至今也无法解决。 |
A******y
发帖数: 2041 | 5 Just because it is commercial primer, does not mean is good if you didn't
check it in your system. Bio-rad should replace the real-time unit, if it
is the problem. |
v***2
发帖数: 131 | 6 Hi, have you run minus RT? and have you checked the primer dimer? I dislike
the BioRad instrument too, it is not as stable as the Applied Biosystem |
A******y
发帖数: 2041 | 7 I used both, AP's new one has software problem, when it crashes you lost the
entire run. The Bio-rad one still have the best software and hardware
setting. I don't see things evaporate from mine.
dislike
【在 v***2 的大作中提到】
: Hi, have you run minus RT? and have you checked the primer dimer? I dislike
: the BioRad instrument too, it is not as stable as the Applied Biosystem
|
o**d
发帖数: 3080 | 8 我们这里前有台CFX96，也是盖子坏了，现在在返厂修理，前期的症状和你遇到的情况
一样，边缘的孔被蒸空了。
和bio－rad好好沟通一下，让他们换修。 |
M*****n
发帖数: 16729 | 9 demo至少应该40% off。
才便宜15% 太亏了。 |
l********s
发帖数: 70 | 10 1 你的总体系别做太小了，至少保证15ul以上，
2 我感觉其实塑料管好于那个贴膜的plate，那个多少会有些leaking |
|
|
m*p
发帖数: 226 | 11 Mine CFX-96 costs 25,000, brand new, works well.
is
【在 s**u 的大作中提到】
: We use Applied Biosystem MicroRNA commericial primer, the primer are all
: good.
: We just find solution evaporation on 2 plates on 8/29/11, one is FAM, one is
: SYRB plate, and it never happened before although I run more than 100
: plates using IQ5 and 30 plates using CFX-96.
: 还有这台CFX-96是作为公司demo用的，原价29，000， 因为作为demo用了半年，所以价
: 格为25，000，我们7月1日才刚刚购买拿到仪器。7月14日才刚刚完成培训。结果2-3个
: 星期就发现问题，至今也无法解决。
|
x**e
发帖数: 163 | 12 我上次在版上也问了同样的问题。和你们一模一样的仪器，我也是很多次（但不是每一
次）做技术重复间的差异都非常大，3个技术重复给的Ct值有1-2的差异。我没有仔细检
查样品蒸发的问题，目测反应后的体积没有发现明显的差异。下次也应该仔细查看样品
的蒸发情况。
看了你的情况，我也不由得怀疑起是不是这个仪器的问题了。 |
s**u
发帖数: 9035 | 13 昨天下午，我做了一块，发现又有更加奇怪的现象发生，96 well Plate右边一半没有
加任何sample, 完全空白，竟然也会出现大量读数，而不是Ｎ／Ａ。
我们老板很生气，决定向Biorad公司交涉更换一台。要到下星期二上班后发email，把
最近一个星期的奇怪现象再次反映给Biorad |
A******y
发帖数: 2041 | 14 What's your plate? I am just curious. It does look like is your instrument.
【在 s**u 的大作中提到】
: 昨天下午，我做了一块，发现又有更加奇怪的现象发生，96 well Plate右边一半没有
: 加任何sample, 完全空白，竟然也会出现大量读数，而不是Ｎ／Ａ。
: 我们老板很生气，决定向Biorad公司交涉更换一台。要到下星期二上班后发email，把
: 最近一个星期的奇怪现象再次反映给Biorad
|
s**u
发帖数: 9035 | 15 We use Biorad multiplate PCR plates Low 96-well clear (MLL9601, LOT# LCRN1,
DOM: 230311), and PCR sealer Microseal 'B' film (MSB1001, LOT# 115084, DOM:
230211) to do our experiment. |
A******y
发帖数: 2041 | 16 Interesting. Did you check your heating element to see if it is clean? For
all RT-PCR,I will recommend semi-skirt white plate to eliminate potential
plate flex and maximum signal. |
s**u
发帖数: 9035 | 17 Bioread sales representative came to our Lab this morning and told us that
Biorad will replace our current CFX96 and replace a new one, hopefully we
will get it next week. |
s**u
发帖数: 9035 | 18 I just got email from FEDEX, our new biorad CFX96 realtime PCR has been
picked up in CA, on 9/9/11 16:19 pm (Eastern time 19:19). |
s**u
发帖数: 9035 | 19 Thanks for your suggestion, from 9/12/11 we started using this kind of plate
, instrument runs well
No edge effects happened, no evaporation happened again.
For
【在 A******y 的大作中提到】
: Interesting. Did you check your heating element to see if it is clean? For
: all RT-PCR,I will recommend semi-skirt white plate to eliminate potential
: plate flex and maximum signal.
|
