h**********r
发帖数: 671 | 1 最近做一个克隆（NdeI/EcoRI），PCR验证是好好的。用PstI验证也正确。可就是用
NdeI/EcoRI酶切不对。
载体和片段都比预期的要大。而且不知道为啥弥散。
麻烦大家帮忙看一下图吧。多谢！ |
H****N
发帖数: 997 | 2 heat your sample at 65 degree for 10 min, then load your sample. If it works
, i will tell you why. |
h**********r
发帖数: 671 | 3 protein binds with DNA? I will try~~
If it works, baozi will be sent to you.
Thanks! |
h**********r
发帖数: 671 | |
H****N
发帖数: 997 | 5 Some REs stick to the DNA after digestion, resulting in the smeared bands
with incorrect migration rates. Heating disrupts the interaction. Heating at
70 degrees could have worked better. I am glad it has worked. Thanks for
the BAOZI. |
b******s
发帖数: 1089 | 6 smear很经常是由于你之前提质粒时不够干净，其中残留的DNase在你加了digestion
buffer后部分降解DNA，前面有人说heat到65度就是为了deactivate DNase。你也可以
用柱子纯化。
size不对不要心怀侥幸。肯定有问题。你还是去sequence一下为好。
【在 h**********r 的大作中提到】
: protein binds with DNA? I will try~~
: If it works, baozi will be sent to you.
: Thanks!
|
H****N
发帖数: 997 | 7 He/she digested the DNA first before heating to 65 degrees. Also, there is
no smear in the other digestions with different REs, which is clearly against
your argument about DNase. DNAse would make the fragments smaller, not bigger.
【在 b******s 的大作中提到】
: smear很经常是由于你之前提质粒时不够干净，其中残留的DNase在你加了digestion
: buffer后部分降解DNA，前面有人说heat到65度就是为了deactivate DNase。你也可以
: 用柱子纯化。
: size不对不要心怀侥幸。肯定有问题。你还是去sequence一下为好。
|
h**********r
发帖数: 671 | |
