g*******8
发帖数: 6 | 1 既然可以用pcr product和primer直接做sequencing，那为什么有时还要用TOPO
cloning先clone，然后再做RCA， 最后用RCA product 和primer来做sequencing呢？
多谢指教！ |
n***w
发帖数: 2405 | 2 PCR product is a heterogenous population which contains many copies of DNA.
Sequencing may give you false positive result. BTW, if you just send PCR
product and primers for sequencing, you cannot get full length (you will
lose ~60 at both ends.)
But after TOPO cloning, it only contains single copy of DNA. |
g*******8
发帖数: 6 | 3 Thanks so much for your reply!
But if so, why most time we still do core sequencing (directly use pcr
product and primer to do sequencing)? Could you please tell me when should
we do core sequencing and when should we do TOPO cloning and then sequencing
?
Thanks again! |
n***w
发帖数: 2405 | 4 Well, I am not an expert on this.
I think it depends on your goal.
Let's say, if I wanna express some protein, then I have to make sure that my
sequence is 100% right to avoid any mutation.
But if you just want to make sure you have a right thing after several round
of PCR, you can just roughly sequence the PCR product.
I hope more people can give input into this post.
should
sequencing
【在 g*******8 的大作中提到】
: Thanks so much for your reply!
: But if so, why most time we still do core sequencing (directly use pcr
: product and primer to do sequencing)? Could you please tell me when should
: we do core sequencing and when should we do TOPO cloning and then sequencing
: ?
: Thanks again!
|
g*******8
发帖数: 6 | 5 You do help me a lot! Thank you! |
G***y
发帖数: 1082 | 6 If you use the TOPO kit, it's ~10$ per reaction and ~a day and half's extra
time. It roughly doubles your cost and time for each sample. You have to
decide if it's worth the trouble/money based on your experiment.
should
sequencing
【在 g*******8 的大作中提到】
: Thanks so much for your reply!
: But if so, why most time we still do core sequencing (directly use pcr
: product and primer to do sequencing)? Could you please tell me when should
: we do core sequencing and when should we do TOPO cloning and then sequencing
: ?
: Thanks again!
|
R*9
发帖数: 15 | 7 主要取决于你的实验目的。举两个例子：
如果你想做克隆，用来表达蛋白，转基因什么的，当然要先克隆再测序了。因为PCR会
导入很多突变，你只有挑选正确的PCR序列才能进行下游的研究。
如果你用来genotyping,那么PCR产物用来测序比克隆要好，因为PCR是一个混合物，通
常对于一个位点，正确的远远大于变异的，那么当你测序的时候，那个变异的信号就被
覆盖了，只有真正的来自基因组/cDNA的变异（SNP或者突变）不会被覆盖，而且你很容
易通过测序结果看出杂合子来（双峰），要达到这些效果你要是测单克隆的质粒的话要
测好几个，费钱，费时，费力，估计没人会去做。
【在 g*******8 的大作中提到】
: 既然可以用pcr product和primer直接做sequencing，那为什么有时还要用TOPO
: cloning先clone，然后再做RCA， 最后用RCA product 和primer来做sequencing呢？
: 多谢指教！
|
