N****n
发帖数: 294 | 1 I have been having great difficulty doing subcloning. It used to be nothing
but now it is some kind of extreme work. I thought it was because my skill
deteriorated badly. Until I sequence a few clones. All the clones showed
that one of the two oligoes had lost half of the designed length. I thought
it was because the oligo wasn't pured and did a gel purification. To my
surprise, that particular oligo ran at the posistion corresponding to the
short size length compeletly. On the other hand, the other oligo ran
correctly. I have been buying my oligo from IDT all the time. Wasted so much
time. Now I have to find a different company for oligo synthesis.
Can someone recommend an oligo synthesis company that you have good
experience? Thanks very much fo the input. |
p*****3
发帖数: 1168 | |
N****n
发帖数: 294 | 3 idtdna is what I used and had problem with.
【在 p*****3 的大作中提到】
: IDT
: http://www.idtdna.com/site
|
a****d
发帖数: 1919 | 4 you may try genescript or genewiz |
m****M
发帖数: 360 | |
m****M
发帖数: 360 | 6 多长的引物？怎么看出的少了一半？上面设有不配对的突变？如果老是这条有问题，而
另外一个是好的话，肯定是你自己的引物和模板那里不对。我用IDT从来没有什么问题
。 |
N****n
发帖数: 294 | 7 It was only 32 nt. Based on sequencing result, it was short by 15nt. I tried
to purify the oligo on PAGE. It was short as well when compared to similar
length oligo. So I am sure it was short.
I have used idt for many years. If there were problem, I normally order news
one thinking my design had problem until I did sequencing and gel
purification. |
m****M
发帖数: 360 | 8 Can you describe it in detail? If 5' is missed, how did you clone the gene (
T-vector ligation)? If 15nt is missed at 3' how the primer complement with
your DNA plate correctly? If it complemented at right position how did you
know 15nt is missed sine it will be identical to one of your DNA strands?
tried
similar
news
【在 N****n 的大作中提到】
: It was only 32 nt. Based on sequencing result, it was short by 15nt. I tried
: to purify the oligo on PAGE. It was short as well when compared to similar
: length oligo. So I am sure it was short.
: I have used idt for many years. If there were problem, I normally order news
: one thinking my design had problem until I did sequencing and gel
: purification.
|
N****n
发帖数: 294 | 9 A PCR was performed and cloned directly into vecotr by ligation (primer
phosphorylated first). Sequencing indicated that one primer was missing 15nt
from the 5' end. Because the 3' end of the primer is intact, that is why
the PCR worked fine.
(
【在 m****M 的大作中提到】
: Can you describe it in detail? If 5' is missed, how did you clone the gene (
: T-vector ligation)? If 15nt is missed at 3' how the primer complement with
: your DNA plate correctly? If it complemented at right position how did you
: know 15nt is missed sine it will be identical to one of your DNA strands?
:
: tried
: similar
: news
|
