发信人: leohawk (leohawk), 信区: Biology
标 题: Re: question about cloning from BAC
发信站: The unknown SPACE (Sun Jul 29 15:28:08 2001), 转信

how about do southern to get physical restriction map(if you have probe for the
gene you want?)

Actually the question you asked is weird, to me :)

if you want to do funsion construct for expression, should you use cDNA??? why
you bother to go after BAC, I think they are all genomic DNA

for expression, the best system I ever used is the PET30(a,b,c) system, all my
proteins got very strong expression, HIS6 helps to purify protein using Ni+
column, thrombin can be used to remove the fursion part from the vector.

if you want to use those proteins for antibody induction, I strongly suggest you
to make tagged transgenes, as antibody doesn't work a lot of times, while
tagged transgene sometimes works very well. To be safe, make both N and C terminal
tagged version.-- if you want to know the localization of your protein in vivo.




【 在 golem (毛毛雄东方必败) 的大作中提到: 】
: 在下手头有几个BAC，想用cut and ligate作几个表达的fusion
: construct.
: 各位能给些建议吗？比如：
: BAC只有在TE里才溶解充分，可是会影响到enzyme digestion.
: gel extraction可能有些麻烦，20kb的片段呀。
: 用什么vector好呢？因为ligation efficiency非常低，该如何设计
: 呢？
: 该如何有效的检验clone序列是否有错？在下手边只有一个基因的mut
: ant可以作suppression.
: 多谢大家发言。


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