v***a 发帖数: 1242 | 1 transfection的efficiency是否跟cell density相关呢?细胞少了是不是efficiency就
高,但是toxicity会大?
我做plasmid DNA transfection的时候,发现普通control(没有加任何transfection
reagent的)和有vector plasmid transfection的control相比,我要观测的蛋白表达
不一样,有vector的蛋白少了很多。不知道这是怎么回事呢?
谢谢~ |
n********k 发帖数: 2818 | 2 it is not a proper comparison, many transfection reagents impact on cells...
think about it, they are peneratring
cell membranes...
transfection
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : transfection的efficiency是否跟cell density相关呢?细胞少了是不是efficiency就 : 高,但是toxicity会大? : 我做plasmid DNA transfection的时候,发现普通control(没有加任何transfection : reagent的)和有vector plasmid transfection的control相比,我要观测的蛋白表达 : 不一样,有vector的蛋白少了很多。不知道这是怎么回事呢? : 谢谢~
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v***a 发帖数: 1242 | 3 嗯,加了transfection reagent的control某些指标明显跟未加reagent的control不一
样了。那我在paper里可以这样说明吗?我老板说应该保持前后一致。比如我前面一个
蛋白在无transfection的cells里表达量有较多,但现在在有vector的control里,蛋白
的表达量下降了好多,用RT-PCR检测几乎就要看不到了;老板就要我重做,说这2个
control非得match。大家遇到过这样的情况吗?如果前后不match,该如何说明比较好?
..
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : it is not a proper comparison, many transfection reagents impact on cells... : think about it, they are peneratring : cell membranes... : : transfection
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c**n 发帖数: 73 | 4 You are supposed to transfect cells with empty vector as a control
好?
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 嗯,加了transfection reagent的control某些指标明显跟未加reagent的control不一 : 样了。那我在paper里可以这样说明吗?我老板说应该保持前后一致。比如我前面一个 : 蛋白在无transfection的cells里表达量有较多,但现在在有vector的control里,蛋白 : 的表达量下降了好多,用RT-PCR检测几乎就要看不到了;老板就要我重做,说这2个 : control非得match。大家遇到过这样的情况吗?如果前后不match,该如何说明比较好? : : ..
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v***a 发帖数: 1242 | 5 Yes, I did. But my boss required that the result from empty vector control
be the same as the normal control to keep the consistence... How can I
explain it solidly enough to convince my boss? Or in fact I did something
wrong that the influence of the empty vector control should not be that much
(made the mRNA level of my protein nearly undetectable)?
【在 c**n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : You are supposed to transfect cells with empty vector as a control : : 好?
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n********k 发帖数: 2818 | 6 your boss is typically those ...I don;t know what to say...BTW, the level of
mRNA etc we normally detect is
always in a relative sense, I don't see either you or your boss has a point
here..., so just do another control to
see whether it is your vector or transfection reagent problems: add
transfections reagents without any DNA or
different vectors...BTW, No offense, I suppose you are a newbie, otherwise u
got quite some work to do with
your design and interpretation of experiments and troublin
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : Yes, I did. But my boss required that the result from empty vector control : be the same as the normal control to keep the consistence... How can I : explain it solidly enough to convince my boss? Or in fact I did something : wrong that the influence of the empty vector control should not be that much : (made the mRNA level of my protein nearly undetectable)?
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g*****y 发帖数: 6325 | 7 cell density在70%-80%的transfection的efficiency 最高
transfection
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : transfection的efficiency是否跟cell density相关呢?细胞少了是不是efficiency就 : 高,但是toxicity会大? : 我做plasmid DNA transfection的时候,发现普通control(没有加任何transfection : reagent的)和有vector plasmid transfection的control相比,我要观测的蛋白表达 : 不一样,有vector的蛋白少了很多。不知道这是怎么回事呢? : 谢谢~
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v***a 发帖数: 1242 | 8 谢谢!!!我承认你说得都对,I've got a long, long way to go
of
point
u
【在 n********k 的大作中提到】![](/moin_static193/solenoid/img/up.png) : your boss is typically those ...I don;t know what to say...BTW, the level of : mRNA etc we normally detect is : always in a relative sense, I don't see either you or your boss has a point : here..., so just do another control to : see whether it is your vector or transfection reagent problems: add : transfections reagents without any DNA or : different vectors...BTW, No offense, I suppose you are a newbie, otherwise u : got quite some work to do with : your design and interpretation of experiments and troublin
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