发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: northern blotting problem
发信站: The unknown SPACE (Tue May 20 18:57:33 2003), 站内信件

my point is to make sure that you have transfer of good
quality and quantity of RNA onto the blotting material.
the washing stringency for your experiment is not quite
high (i.e., 42C). i think the problem is mainly due to
bad RNA or probe. if there is a lot of radioactive
nucleotides incorporated into the probe, you should
detect very hot signal after purification. denature the
probe before use. GAPDH is a housekeeping gene that is
typically used for normalization of load. i suggest you
read a northern protocol from Ambion.
http://www.ambion.com/techlib/prot/bp_1940.pdf
hope it solves your problem.

【 在 gooddream (damn, damn, it is not right) 的大作中提到: 】
: I didn't stain the blot after transfer and used DNA as probe. Also,I washed
: the membrane at 42C by (2X SSPE and 0.1%SDS) twice,total 20mins and (0.5XSSPE
: and 0.1%SDS)twice,total 20 mins. DId that hurt the blot?
: By the way,what is GAPDH? And what is the purpose to stain the blot with
: methyl green after transfer? Sorry,some simple questions.
: Thanks a lot!
: 【 在 Marble (小石头哥哥) 的大作中提到: 】
: : i would like to know whether you stain the blot with methyl
: : green after transfer; also, do you use cRNA or DNA as probe?
: : what is the hyb condition, particularly the temperature and
: : buffer? wash with increasing SDS and decreasing salt may
: : strip some of the signal sometimes. i strongly recommend you
: : check everything before you restart it. maybe you should try
: : northern with GAPDH first to check the quality of the blot
: : and your technique. northern blot is difficult, yet i think
: : if you can make sure of the quality of the RNA, efficiency
: : of transfer, quality of the probe, you should get something.
: less
: think
: details?


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