w******e
发帖数: 1187 | 1 suppose I have a pool of oligos (say 100 unique sequences), each
15-20base long, biotinylated on 5' end. what's the best way
to sequence them?
I guess if w/o biotin, I can probably make ds using random primer, and then
clone into sequence vector. can I do sth similar with biotinylated oligos?
thx!! |
c*****g
发帖数: 66 | 2 我没做过这个实验。概念上说，你经过一轮pcr (在primer上加universal adaptor)，
biotin就被稀释了一倍，你多几个循环，就可以克隆了。
then
【在 w******e 的大作中提到】
: suppose I have a pool of oligos (say 100 unique sequences), each
: 15-20base long, biotinylated on 5' end. what's the best way
: to sequence them?
: I guess if w/o biotin, I can probably make ds using random primer, and then
: clone into sequence vector. can I do sth similar with biotinylated oligos?
: thx!!
|
s******y
发帖数: 28562 | 3 直接送去做质谱应该是最简单的方法吧？
then
【在 w******e 的大作中提到】
: suppose I have a pool of oligos (say 100 unique sequences), each
: 15-20base long, biotinylated on 5' end. what's the best way
: to sequence them?
: I guess if w/o biotin, I can probably make ds using random primer, and then
: clone into sequence vector. can I do sth similar with biotinylated oligos?
: thx!!
|
w******e
发帖数: 1187 | 4 didn't know that's an option... do you know how much would it cost? is it
possible to delineate all the hundreds of different sequences together?
all of them are made of ATCG, so I would guess different sequence could
have the same mass but different sequence?
thx!
【在 s******y 的大作中提到】
: 直接送去做质谱应该是最简单的方法吧？
:
: then
|
w******e
发帖数: 1187 | 5 I don't know how to do PCR in this case, as I have 0 idea what the sequence
is (so no primer available on either end).
I meant to use the random primer to do one round of primer extension. Maybe
I can add some tag to the random primer to enable PCR? any thoughts?
【在 c*****g 的大作中提到】
: 我没做过这个实验。概念上说，你经过一轮pcr (在primer上加universal adaptor)，
: biotin就被稀释了一倍，你多几个循环，就可以克隆了。
:
: then
|
s******y
发帖数: 28562 | 6 可以啊，小菜一碟。
就做microsequencing 好了。告诉他们有几个基团有可能被biotinylated,
让他们search database 的时候记得加上那个option
【在 w******e 的大作中提到】
: didn't know that's an option... do you know how much would it cost? is it
: possible to delineate all the hundreds of different sequences together?
: all of them are made of ATCG, so I would guess different sequence could
: have the same mass but different sequence?
: thx!
|
w******e
发帖数: 1187 | 7 naive question -- can MS distinguish two oligos with same base composition
but different sequence? I have little idea how MS works...
【在 s******y 的大作中提到】
: 可以啊，小菜一碟。
: 就做microsequencing 好了。告诉他们有几个基团有可能被biotinylated,
: 让他们search database 的时候记得加上那个option
|
s******y
发帖数: 28562 | 8 可以，用microsequencing 完全可以。那个是他们的基本技术之一。
具体让质谱的人来说吧。
【在 w******e 的大作中提到】
: naive question -- can MS distinguish two oligos with same base composition
: but different sequence? I have little idea how MS works...
|
w******e
发帖数: 1187 | 9 thx a lot!! you are the best~
【在 s******y 的大作中提到】
: 可以，用microsequencing 完全可以。那个是他们的基本技术之一。
: 具体让质谱的人来说吧。
|
R****n
发帖数: 708 | 10 Maybe I am wrong. But I don't think that you can use MALDI-TOF for oligo
nucleotide sequencing.
【在 s******y 的大作中提到】
: 可以，用microsequencing 完全可以。那个是他们的基本技术之一。
: 具体让质谱的人来说吧。
|
R****n
发帖数: 708 | 11 If you can remove the biotinylated group, you can build a miRNA library for the 2nd generation sequencing system.
then
【在 w******e 的大作中提到】
: suppose I have a pool of oligos (say 100 unique sequences), each
: 15-20base long, biotinylated on 5' end. what's the best way
: to sequence them?
: I guess if w/o biotin, I can probably make ds using random primer, and then
: clone into sequence vector. can I do sth similar with biotinylated oligos?
: thx!!
|
