请教： dual luciferase assay （luciferase/Renilla） - 未名空间MITBBS历史存档

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请教： dual luciferase assay （luciferase/Renilla）

请教： dual luciferase assay （luciferase/Renilla）# Biology - 生物学

t*n2015-02-28 08:02

1 楼

读数都是个位数或两位数，另外一个实验室的同事说他以前的读数都至少是几千上万的
。我们用的plate reader是BioTek的 Gen 5, reagents from Promega.
这正常么？哪个地方搞错了？非常感谢！

m*D2015-02-28 08:02

2 楼

没用过这种仪器。我们的仪器会有不同的setting: measure的时间往往会不一样的。

【在 t**********n 的大作中提到】

: 读数都是个位数或两位数，另外一个实验室的同事说他以前的读数都至少是几千上万的
: 。我们用的plate reader是BioTek的 Gen 5, reagents from Promega.
: 这正常么？哪个地方搞错了？非常感谢！

t*n2015-02-28 08:02

3 楼

I followed the standard protocol of Promega kit.
Many thanks.

【在 m*********D 的大作中提到】

: 没用过这种仪器。我们的仪器会有不同的setting: measure的时间往往会不一样的。

n*w2015-02-28 08:02

4 楼

For my positive control, I usually get 1000-2000 and the internal control is
about 5-10, so the ratio is always about 200ish.
My other experimental ones, which I didn't expect to have signals, would
give me values of single or double digits and the ratio ranges from 0.5 to 3
,4 ish...
So how about your control? The positive controls, both at your hand and his
should yield similar ratios. If this is okay, then you may not need to worry
much.
Even for negative results, we want interpretable negative results.

h*y2015-02-28 08:02

5 楼

这读数太低了，你转个psic heck-2做个正对照看看,至少要几万，🈶的时候一
百万都有

L*m2015-02-28 08:02

6 楼

我觉得这个得看机器。
很久以前用过不同的两台机器，一个小于1000的书都不可信，一个两位数就很好。你看
看机器的背景读数多少

【在 t**********n 的大作中提到】

t*n2015-02-28 08:02

7 楼

Many thanks.
As for the control, we have a paralled transfection with GFP plasmid, whose
expression is low while positive.
Renilla readouts are 2~4，evan using different amount of plasmid, which is
almost same as the blank(就是96孔板上啥也没有的wells).
Luciferase readouts are 8~12( expected as negative).
We just had another experiment: luciferase readouts ~60, expected as
positive while Renilla readouts 8~11.
Not sure whether I expressed myself clearly or not.

is
3
his
worry

【在 n***w 的大作中提到】

: For my positive control, I usually get 1000-2000 and the internal control is
: about 5-10, so the ratio is always about 200ish.
: My other experimental ones, which I didn't expect to have signals, would
: give me values of single or double digits and the ratio ranges from 0.5 to 3
: ,4 ish...
: So how about your control? The positive controls, both at your hand and his
: should yield similar ratios. If this is okay, then you may not need to worry
: much.
: Even for negative results, we want interpretable negative results.

t*n2015-02-28 08:02

8 楼

非常感谢Leaderham,以及上一帖的hellenny！

【在 L*******m 的大作中提到】

: 我觉得这个得看机器。
: 很久以前用过不同的两台机器，一个小于1000的书都不可信，一个两位数就很好。你看
: 看机器的背景读数多少

t*n2015-02-28 08:02

9 楼

同时还感谢回复的acumen.

【在 t**********n 的大作中提到】

: 非常感谢Leaderham,以及上一帖的hellenny！