Re: 关于genotyping 的几个问题. - 未名空间MITBBS历史存档

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Re: 关于genotyping 的几个问题.

Re: 关于genotyping 的几个问题.# Biology - 生物学

b*t2002-12-10 08:12

1 楼

depending on the sizes of the bands (usually two). if they are both big and
close in size, then you will have to run it really slow for a long time (i
have one 11kb/9kb southern that i run at 40V for 10 hrs!); otherwise, you
can run it much faster (70V, 3 hrs.)
this should have been determined BEFORE you make your transgenic mice.
you should be able to predict the sizes/conditions based on your original
constructs.
depending on your probe. usually two bands: one endogenous, one transgenic.
good

M*e2002-12-10 08:12

2 楼

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 10