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发信人: adeno (天夺其魄), 信区: Biology
标 题: Re: 目前最常用的点突变方法是什么?
发信站: The unknown SPACE (Thu Jun 13 01:32:11 2002) WWW-POST
Good point. OK, this is the best-kept secret of the century, hehe. The truth
is I always do my first round of PCR with Pfu (Pfx from invitrogen seems to be
quite efficient). If not for ease of cloning I will do second round with it
too. But since the fragment you want to mutate is usually around only 1 kb,
taq will be good enough.
Another reason I prefer Pfx in first round is that Taq should introduce an
overhang at the end of the product, sometimes this maks you troube in second
round by introducing insertion
【 在 golem (杯杯狗愣) 的大作中提到: 】
: the caution should come from PCRs. You know, some fragments from 1st PCR
: incorporated errors. These fragments are more suitable for PCR reaction than
: the correct version. So if you use 2-step PCR, chances are the ratio of
wrong
: templates becomes bigger.
:
: 【 在 adeno (天夺其魄) 的大作中提到: 】
: : not really, you have actually same # of steps as 2-step PCR
: :
: : One step: PCR----DpnI-----Self ligation
: : two step: PCR----PCR---TA cloning
: :
: : And actually your steps are more tricky, if you consider that Pfu has low
: : processivity while you have to PCR the full length of the plasmid which is
: at
: : least 3kb, you didn't really save time; and also compared to the
efficiency
: : of 2-step which is 100%, the efficiency of your method is low; another
: : consideration may not be so important to you. you have to buy expensive
kit
: to
: : do this, while 2-step cost almost nothing.
: :
: :
: : 【 在 gzip (好好睡一觉) 的大作中提到: 】
: : : 其实也不必用两步PCR啦。
: : : 偶们用一步PCR，引物设计跟你说的一样，Pfu扩第一步，PCR产物用DpnI切。DpnI
专
: 切
: : 甲
: : : 基化的DNA，
: : : 这样PCR合成的DNA不会被切，而模板被切掉。切完直接转到大肠里就行了。
: : : 在偶手里一般是>80%。在偶领导手里是100%，呵呵。
: : : 一步的好处就不用我说了吧。
: : :
: : : 【 在 akite (找路的人) 的大作中提到: 】
: : : : to my experience, the efficiency is 100%, if you can get both
first-step
: : : : products correctly.
: : : : the trick is the 3' end of the front part and the 5' end of the rear
: part
: : : are
: : : : the same, notice my first sentence, "a pair of COMPLEMENTARY primers",
: so
: : in
: : : : the first annealing round of the 2nd PCR, the two templete DNA
molecules
: : : : anneal, in the first elongation round, both annealed chains elongate
to
: : the
: : : : end and get the full lenth gene. in the next steps, just normal PCR
le.
: : : :
: : : : 【 在 aoexy (lili) 的大作中提到: 】
: : : : : then how about the effciency? How does it happen since the template
: DNA
: : : are
: : : : : two separate one?
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※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.249.150.80]
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