发信人: cDNA (岁月蹉跎), 信区: Biology
标 题: Re: PCR产物酶切一问
发信站: Unknown Space - 未名空间 (Fri Sep 5 05:48:16 2003), 站内信件
first of all, everything could happen in biology.. hehe..
so, for trouble shooting:
1. run gel to check your sample before digestion but after
purification.
OK --> digestion problem
smear --> purification problem.
2. if purification problem, try to use a PCR purification kit.
3. if digestion problem.
A. make sure the fragment you cloned did not contain any
HindIII/EcoRI
B. try a sequencial digestion..
4. just a reminder:
HindIII will have prolbem to cut when your site really close to
the end... it may need up to 6 extra bp to avoid this..
EcoRI is an enzyme contain high star activity...
so frankly speaking, your original cloning stratige is not the best,
if there is other choices.. hehe..
anyway, hope things work out for you... :)
【 在 batuloo (爱育栗拔力八达) 的大作中提到: 】
: 我要把一段基因clone到一个载体里面去
: 方法是用PCR引物将HindIII EcoRI位点引入PCR两头
: 然后粪房抽提 然后沉淀 然后双酶切2个小时
: 然后直接跑胶发现是smear
: PCR产物本来是很好的band
: 这smear到底是怎么回事
: 我相信水 buffer bsa 酶 都是好东东
: 大家可怜可怜我 帮我一把
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※ 来源:.Unknown Space - 未名空间 mitbbs.com.[FROM: 64.160.]
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