发信人: Ras (很颓的猪。), 信区: Biology
标 题: Re: who makes the Competent cell called NM522?
发信站: Unknown Space - 未名空间 (Mon Mar 8 17:42:56 2004), 转信

ft. 你们用英文是讲不清楚了, 要不就是相互都没pay attention好好看。

GENESIS的意思是不管你用多少细胞，如果你的细胞好，antibiotics质量也好，
ligation做得也clean,那你negative control就应该很clean, 没什么clonie.
跟用了多少细胞没关系。同时ligation plates里应该有大量colonies而且
false positive的比例也应该很少。所以没必要降低细胞量。也没必要因为
clonies多而多做miniprep.

除非你切了后的vector超级sticky, 而且CIP不干净,
导致self-ligation很多（这句偶加的，呵呵）。

当然偶自己做cloning很烂，经常ligation plate才长一个，
一挑还真有insert. 哈哈，倒是省miniprep.

【 在 nohate (无恨) 的大作中提到: 】
: I guess you still don't understand what I am talking about, it is either I
: didn't make it clear, or you just didn't pay attention.
: To reduce the cell used in the transformation means reduce both the control
: and the one with plasmid. For example, if 50 microliter cell give like 20
: colonies in the negtive control plate, and 25 in the ligation plate, if we use
: 20 microliter cell in both transformation, maybe it will end up with 5
: colonies in negtive control and 10 in ligation plate, will that help in regard
: of how many colonies we should pick to do minipreps?
: 【 在 Genesis (LIH) 的大作中提到: 】
: : ok, enough for kidding. here is point: if you are using the right amount
: : of antibiotics, and the antibiotics is good not degraded, you should NOT
: : see ANY colonies on cell only plates, assuming you are not putting tons
: : of bacteria on it. and even if you put a lot on the plate and there is
: : some growth, they should not be distinctive and separatable colonies, more
: : likely you would get sth look like confluent growth.
: : the amount of the cell you use in a control plate should not be a factor at
: all.
: : if it appeares to be factor, then something is wrong with your experiment


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※ 修改:．Ras 于 Mar 8 17:44:57 修改本文．[FROM: 24.136.]
※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 24.136.]