PCR std dev 总是太高。。。# Biology - 生物学
d*i
1 楼
三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
大家都是怎么set up的？谢谢！
大家都是怎么set up的？谢谢！
n*w
2 楼
搭车同问。
k*l
3 楼
mix 包括模板吗？
我一般不包括，然后单独加模板
一定要混匀，尽量不要打出气泡
【在 d*****i 的大作中提到】
: 三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
: 大家都是怎么set up的？谢谢！
我一般不包括，然后单独加模板
一定要混匀，尽量不要打出气泡
【在 d*****i 的大作中提到】
: 三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
: 大家都是怎么set up的？谢谢！
A*y
4 楼
Mix master mix including everything except the template. Pipet into each
well, then add the template cDNA or mRNA. All procedures are on ice. The
plate is on a cooler plate holder. My lab also uses a automatic repeater
Eppendorf Stream for the addtion.
Also, when is last time your get your pipetor a calibration?
【在 d*****i 的大作中提到】
: 三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
: 大家都是怎么set up的？谢谢！
well, then add the template cDNA or mRNA. All procedures are on ice. The
plate is on a cooler plate holder. My lab also uses a automatic repeater
Eppendorf Stream for the addtion.
Also, when is last time your get your pipetor a calibration?
【在 d*****i 的大作中提到】
: 三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
: 大家都是怎么set up的？谢谢！
s*s
5 楼
这个和calibration一点关系都没有，就算都不准，只要consistent
就应该一样。有可能是有气泡了，还一个可能是machine加热不均匀。
有钱的用一样的样品铺满96孔板三次，看一下每个孔的差别多大
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
就应该一样。有可能是有气泡了，还一个可能是machine加热不均匀。
有钱的用一样的样品铺满96孔板三次，看一下每个孔的差别多大
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
A*y
6 楼
Palmface! There are two kinds of off calibration. One is the measurement
is off. The other is inconsistancy from one additon to another. The bubble
can be eliminated becuase you should spin down the plate before the run.
Although, the uneven heating is also very likely, then it will be semi-
costly to repair. Btw, does your instrument require an internal standard or
not (i.e. ROX dye etc.)?
【在 s******s 的大作中提到】
: 这个和calibration一点关系都没有，就算都不准，只要consistent
: 就应该一样。有可能是有气泡了，还一个可能是machine加热不均匀。
: 有钱的用一样的样品铺满96孔板三次，看一下每个孔的差别多大
is off. The other is inconsistancy from one additon to another. The bubble
can be eliminated becuase you should spin down the plate before the run.
Although, the uneven heating is also very likely, then it will be semi-
costly to repair. Btw, does your instrument require an internal standard or
not (i.e. ROX dye etc.)?
【在 s******s 的大作中提到】
: 这个和calibration一点关系都没有，就算都不准，只要consistent
: 就应该一样。有可能是有气泡了，还一个可能是machine加热不均匀。
: 有钱的用一样的样品铺满96孔板三次，看一下每个孔的差别多大
l*s
7 楼
很可能是引物浓度不够高，造成扩增效率不同所致。正常是200-500nm，最后扩增曲线
达到同样饱和平台高度。PCR REPLICATE 之间正常的误差范围是0.0-0.2 cycle,超过0.
5 cycle就是大问题了。
【在 d*****i 的大作中提到】
: 三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
: 大家都是怎么set up的？谢谢！
达到同样饱和平台高度。PCR REPLICATE 之间正常的误差范围是0.0-0.2 cycle,超过0.
5 cycle就是大问题了。
【在 d*****i 的大作中提到】
: 三个replicates，大部分时候都提示sd大于0.5。我是先配好mix，直接加入vival里。
: 大家都是怎么set up的？谢谢！
d*i
8 楼
实验室的pipet都是一年calibrate一次吧
bubble
or
【在 A******y 的大作中提到】
: Palmface! There are two kinds of off calibration. One is the measurement
: is off. The other is inconsistancy from one additon to another. The bubble
: can be eliminated becuase you should spin down the plate before the run.
: Although, the uneven heating is also very likely, then it will be semi-
: costly to repair. Btw, does your instrument require an internal standard or
: not (i.e. ROX dye etc.)?
bubble
or
【在 A******y 的大作中提到】
: Palmface! There are two kinds of off calibration. One is the measurement
: is off. The other is inconsistancy from one additon to another. The bubble
: can be eliminated becuase you should spin down the plate before the run.
: Although, the uneven heating is also very likely, then it will be semi-
: costly to repair. Btw, does your instrument require an internal standard or
: not (i.e. ROX dye etc.)?
d*i
9 楼
包括。气泡怎么避免啊？
【在 k****l 的大作中提到】
: mix 包括模板吗？
: 我一般不包括，然后单独加模板
: 一定要混匀，尽量不要打出气泡
【在 k****l 的大作中提到】
: mix 包括模板吗？
: 我一般不包括，然后单独加模板
: 一定要混匀，尽量不要打出气泡
d*i
10 楼
我的master mix加了template。为啥要分开加template啊？分开加的话20uL总体积cDNA
加多少ng及体积？
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
加多少ng及体积？
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
A*y
11 楼
Our lab uses 23 uL of master mix per well then adds 2 uL of 50 ng of mRNA.
To eliminate bubbles, you have to spin your plate before putting it into the
instruments. Wait, so your three replicates are from the same master mix
with templates in them i.e. you just add exactly the same solution to 3
different wells? You should have minimum errors. In this case, it could be
your instrument.
cDNA
【在 d*****i 的大作中提到】
: 我的master mix加了template。为啥要分开加template啊？分开加的话20uL总体积cDNA
: 加多少ng及体积？
To eliminate bubbles, you have to spin your plate before putting it into the
instruments. Wait, so your three replicates are from the same master mix
with templates in them i.e. you just add exactly the same solution to 3
different wells? You should have minimum errors. In this case, it could be
your instrument.
cDNA
【在 d*****i 的大作中提到】
: 我的master mix加了template。为啥要分开加template啊？分开加的话20uL总体积cDNA
: 加多少ng及体积？
d*i
12 楼
一般测之前2000rpm spin 1min。但是拿出来后好像还是有bubble，不在well底下，在
上面。
那台ABI是系里公用的，看实验室其他人做的结果也都很好，估计不是仪器问题。
mRNA RT后还测cDNA浓度吗？我一般是测cDNA浓度，然后一个well里加100ng cDNA。
primer浓度一般用多少呢？
the
be
【在 A******y 的大作中提到】
: Our lab uses 23 uL of master mix per well then adds 2 uL of 50 ng of mRNA.
: To eliminate bubbles, you have to spin your plate before putting it into the
: instruments. Wait, so your three replicates are from the same master mix
: with templates in them i.e. you just add exactly the same solution to 3
: different wells? You should have minimum errors. In this case, it could be
: your instrument.
:
: cDNA
上面。
那台ABI是系里公用的，看实验室其他人做的结果也都很好，估计不是仪器问题。
mRNA RT后还测cDNA浓度吗？我一般是测cDNA浓度，然后一个well里加100ng cDNA。
primer浓度一般用多少呢？
the
be
【在 A******y 的大作中提到】
: Our lab uses 23 uL of master mix per well then adds 2 uL of 50 ng of mRNA.
: To eliminate bubbles, you have to spin your plate before putting it into the
: instruments. Wait, so your three replicates are from the same master mix
: with templates in them i.e. you just add exactly the same solution to 3
: different wells? You should have minimum errors. In this case, it could be
: your instrument.
:
: cDNA
A*y
13 楼
If I remeber correctly, it is 200nM for primers. However, if everyone in
your lab does not have your problem...I hate to say it, it is YOU. I have
meet people can not use pipettor properly before.
your lab does not have your problem...I hate to say it, it is YOU. I have
meet people can not use pipettor properly before.
z*6
14 楼
大家觉得气泡真会影响结果吗？难道不应该被煮出去吗？... 我一直不明白，虽然我每
次上机前都3-4000 rpm离一下心，但是bubble有时候还在... 貌似室温离就能消除气泡
，但是我真的怀疑气泡到底对最后结果有多大影响...
我每次内参，比如GAPDH这种，triplicates重复性非常高，也就差0.0几... 但是我的
gene of interest variation也会到0.5，而且我是做老鼠器官的，反正肯定要用n只老
鼠才能看到2倍左右的差别，triplicates重复这样我也就无所谓了...
【在 d*****i 的大作中提到】
: 包括。气泡怎么避免啊？
次上机前都3-4000 rpm离一下心，但是bubble有时候还在... 貌似室温离就能消除气泡
，但是我真的怀疑气泡到底对最后结果有多大影响...
我每次内参，比如GAPDH这种，triplicates重复性非常高，也就差0.0几... 但是我的
gene of interest variation也会到0.5，而且我是做老鼠器官的，反正肯定要用n只老
鼠才能看到2倍左右的差别，triplicates重复这样我也就无所谓了...
【在 d*****i 的大作中提到】
: 包括。气泡怎么避免啊？
z*6
15 楼
对了，我现在都用SYBR green 10ul的体系... 也没什么问题...
z*6
16 楼
我到最后一步加cDNA模板时，都用排枪加2 ul... 这个variation本身估计就不小...
但是我现在ct variation只要在0.5以内，我都认了... 尤其是GAPDH没什么variation
的情况下... 就是我的基因比较tricky而已...
但是我现在ct variation只要在0.5以内，我都认了... 尤其是GAPDH没什么variation
的情况下... 就是我的基因比较tricky而已...
S*9
17 楼
Can you give me a link for automatic repeater Eppendorf Stream?
Thanks
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
Thanks
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
z*8
18 楼
如果仪器老，就是仪器问题。 如果是新仪器，就要改善加法了。
z*6
19 楼
Thanks for your helpful info. I just realized that our lab has several
Eppendorf repeaters. I could just buy some combitips to add master mix.
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
Eppendorf repeaters. I could just buy some combitips to add master mix.
【在 A******y 的大作中提到】
: Mix master mix including everything except the template. Pipet into each
: well, then add the template cDNA or mRNA. All procedures are on ice. The
: plate is on a cooler plate holder. My lab also uses a automatic repeater
: Eppendorf Stream for the addtion.
: Also, when is last time your get your pipetor a calibration?
y*a
20 楼
你PCR tube的盖子厚度均一吗？或者那个盖子是专门做real-time的吗？我以前遇到过
相同问题，后来发现盖子用错了...
相同问题，后来发现盖子用错了...
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