发信人: moso (野鬼), 信区: Biology
标 题: Re: PCR 问题 - single-sided PCR
发信站: BBS 未名空间站 (Wed Sep 21 18:42:47 2005), 转信
I GUESS THERE ARE TWO CONCERNS YOU NEED TO IDENTIFY BEFORE THE FURTHER STEP TO
OPTIMIZE YOUR PROTOCOL.
1. IF THERE IS SELF-PRIMER EXISTING IN YOUR SYSTEM. YOU NEED TO CHECK BOTH
TEMPLATE AND PRIMER TO SEE IF THERE POSSIBLY IS UNEXPECTED PRIMER. IF YES, YOU
MAY HAVE TO CHANGE PRIMER OR OPTIMIZE YOUR PCR PROTOCOL TO AVOID THE
UNEXPECTED
AMPLIFICATION.
2. IF THE REAGENTS YOU USED ARE ALL CLEAN. SET UP SOME CONTROLS TO RULE OUT
ANY OF THOSE CONTAMINATED REAGENTS.
【 在 cyclodextrin (cyclo) 的大作中提到: 】
: 想知道这里有没有人有做 single-sided PCR 来合成 single-stranded DNA 的经验.
: 我的目标是从一个大约300bp的DNA (double-stranded, gel purified) 为template,
只
: 加入一个大约20-mer的primer, 做出大约250 nt的single stranded DNA. 但是我做出
来
: 的结果总是double-stranded DNA (能被restriction enzyme 切)而且根据跑胶,大小
也
: 是300bp的样子。这样的结果很confusing, 因为只加了一个primer, 为什么出来double
: strand? 如果是可能的,怎么样改进才能得到single strand呢?
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