发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: Southern Blot问题求教！
发信站: The unknown SPACE (Sun Mar 2 18:53:29 2003), 站内信件

for genomice southern, you should have very good quality DNA
and digest it completely. it is better to purify the gDNA
and remove anything that may inhibit digestion. typically,
i would digest about 10-20ug of gDNA O/N with enough enzyme
and add another shot next day. it is not suggested to add
EtBr into the gel, though i found it was fine with mine.
you should follow a good protocol that is known to work for
genomic southern. my opinion is that transfer should be very
good. after depurination and denaturation, use 10X or 20X SSC
for transfer, use nylon membrane (it is pretty strong for
intensive wash, i use Zeta membrane). i think you should
have a good control for southern blot, probably probe the
filter for another gene. the quality of the probe is really
important, and i suggest you try a couple of different
cDNA probe or genomic probe, better do a BLAST 1st. and it
would be good if you can actually load some cDNA (cloned,
1ng more than enough) as ctrl. btw, signal from DNA ladder
is not surprising, since there is hyb. b/w intronic DNA
and ladder DNA, which is probably from lambda phage (thus
i assume you use gDNA probe). finally, the database is not
completely accurate, and there could be restriction
polymorphism for different strains (mice?)


【 在 smallbite (小咬) 的大作中提到: 】
: 欲用Southern Blot检测mouse genomic DNA的EcoRI酶切片断
: 从网上数据库分析应该可以检测到10.2k band
: probe为1kb PCR片断，经测序与mouse genomic数据库中的sequence完全吻合
: 且正位于10.2k EcoRI酶切片断之中
: 实验结果却令人十分失望，没有看到任何清晰的band，32P显影呈smear状，基本
: 与DNA胶EB染色形状符合，仅smear最上端（大约>20kb)地方似乎有带
: 个人觉得原因可能有如下几条：
: 一. probe specificity很差，与所有DNA均杂交，事实上DNA ladder确实也杂交上
: 了（可是1kb，sequence 100% 吻合呀？这样还不够？I 服了 U！）
: 二. 实验技术问题，没杂交好，或洗得不够，可是加大wash强度显影仍是脏脏的smear
: 没看到10k的地方有出现band的迹象
: （另，按照molecular clone上的说法，wash完后membrane用geige counter探
: 测，没杂交的地方应该没有反应，我的可是嘎啦嘎啦直响，怎么洗也一样，另外
: 据说single copy的gene杂交后也count不到，是这样的吗？）
: 三. 网上Database中的sequence有问题，根本就没有10.2k的EcoRI片断，而是非常大
: 的片断，所以我看到smear最上端似乎有带。
: 另外，网上这段sequence中实际上有100个连续的N，请问这100个N代表这地方
: 确实有100个bp，但序列未知呢？还是这儿有一段序列未知，具体是多少个bp
: 也是不清楚？
: 急盼有经验的同志指点，这个实验对我比较重要，做不出来最近很是着急上火，唉！


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※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.118.]