发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 关于genotyping 的几个问题.
发信站: The unknown SPACE (Fri Jan 3 17:08:20 2003), 站内信件

refer to NAR 1991 19:42 P.W.Laird et al
i have tried to optimize the condition and it works pretty
well for me. basically, cut ~5mm tail when the mickeys are
about 14-d-old (do not forget ear tag), if you worry about
cross-contamination, clean scissors with 70% ethanol and
flame, otherwise, i typically clean it after collecting 5
samples. digest the tail in a tightly sealed epp. with 500ul
lysis buffer, 5ul proteinase K (10mg/ml). rotate O/N at 55C,
though 6hrs is pretty much sufficient. spin 10min @ maximum,
transfer suppernatant (you will notice sth viscous, that is
DNA, do not discard it!) add 1V 100% ethanol, shake, the DNA
will precipitate. if not too much, you can spin it. otherwise,
use a tip to pick the coiled DNA out and dissolve in 500ul
TE @ 55C 1-2hrs. such DNA is good for PCR or Southern, though
you may further treat it with chloropan to remove protein
contamination.
forget the lysis buffer, it is 100mM Tris-HCl pH8.5, 5mM EDTA,
0.2% SDS, 200mM NaCl
【 在 mrp (mrp) 的大作中提到: 】
: can u poist ur protocol here? Thanks.
: 【 在 Marble (小石头哥哥) 的大作中提到: 】
: : so are you working on "transgenic" or knockout? last time
: : i mentioned about inverse PCR, then i thought that there
: : could be multiple insertions and you do not really know
: : exactly the "effective" expression. so it would be great
: : if the transgene has certain tag for characterization. if
: : you just need to isolate tail DNA for PCR/Southern, i have
: : a good protocol and i can forward to your email. not necessary
: : to get those fancy kits.


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※ 修改:．Marble 于 Jan 3 17:24:53 修改本文．[FROM: 128.118.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.118.]