Re: 求助: Southern Hybridization with olig - 未名空间MITBBS历史存档

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Re: 求助: Southern Hybridization with olig

Re: 求助: Southern Hybridization with olig# Biology - 生物学

m*o2004-08-04 07:08

1 楼

Whether or not the probe is excessive basically depends on the level of the
targetting DNA. Generally I believe the concentration of your probe should be
enough for the detecting because 10ng/ml of a 16mer oligo aproximately equals
to 1.5nM by molar concentration, which could capture 1.5pmol targetting DNA if
it bound at a ratio of 1 to 1.
To label or not to label, it depends on the method you used to detect the
targetting. If you use, for example, biotin to lable to probe, then label the
probe

m*o2004-08-04 07:08

2 楼

I don't quite understand how you label the probe. Usually the methods for
making non-radioactive probe incorporate only a small unit, like a biotin,Dig
or fluorescein into the probe via UTP or others instead of directly incorporate
the enzymes for detection,like HRP, AP. Since it is not easy to keep the full
enzymetic activity during the long experiment procedure.
So if you use klenow to label the probe, you can deactivate the klenow by
heating the reaction at 75C for 20min in presence of 10mM E

s*s2004-08-04 07:08

3 楼

It could be. I do think this method is too sensitive, so it has many critical
parameters (p8 of protocol). If you have one mismatch parameter, the result
screw-up.
I sugest bigfatliar do the dot blot control test first. You just have to drop
several dots of genomic DNA on the membrane, using different concentration.
For example, 1ul of DNA in 10ug/ul, 1ul of DNA in 5ug/ul.... You can have 5
drops in a roll, followed by cross link, hybridization, etc. If this control
membrane worked, then your pr

【在 m**o 的大作中提到】

: I don't quite understand how you label the probe. Usually the methods for
: making non-radioactive probe incorporate only a small unit, like a biotin,Dig
: or fluorescein into the probe via UTP or others instead of directly incorporate
: the enzymes for detection,like HRP, AP. Since it is not easy to keep the full
: enzymetic activity during the long experiment procedure.
: So if you use klenow to label the probe, you can deactivate the klenow by
: heating the reaction at 75C for 20min in presence of 10mM E