B******o
发帖数: 496 | 1 最近花了很多时间折腾RACE。无论是3'还是5'都是铩羽而归。
目前的情况是，3'RACE。 Clontech的kit提供的primer实际上就是Oligo dT加一段
specific的RACE primer。整个反应里就RT酶，total RNA，Oligo dT和dNTP。
cDNA产物用gene specific primer A + RACE primer没有任何条带出来。作为control
，用另一个gene specific的primer B和A配对却有条带。说明RT反应没有问题。可是RT
反应要进行，RACE primer conjugated oligo dT也要能work才行啊？
哪位能说说到底什么地方出问题？ 万分感谢 | c********b
发帖数: 363 | 2 I used Invitrogen GeneRACer kit，which suggested nested-pcr.
Invitrogen's oligo adapter is about 60 nt，first PCR used adapter 5‘-end ~
30 nt primer and gene specific（GS) primer. Then they use close to adapter 3
'-end nested primer and another nested GS primer.
After the nested PCR, I sometimes still see multiple bands. I have to gel-
purify the PCR products with correct size and sometimes to repeat 2nd PCR to
amplify it before cloning.
One suggestion is to purify mRNA for RACE, which gives you much cleaner
result. If not, you can try to use LiCl to precipitate large ribosomal RNAs
before RACE. Also, you can try to use only another GS specific primer+ same
adapter primer for nested PCR before you buy another expensive kit. I used
to the oligo-dT-adapter primer(GCGC repeats) individually instead of buying
a kit.
Good luck!
control
RT
【在 B******o 的大作中提到】
: 最近花了很多时间折腾RACE。无论是3'还是5'都是铩羽而归。
: 目前的情况是，3'RACE。 Clontech的kit提供的primer实际上就是Oligo dT加一段
: specific的RACE primer。整个反应里就RT酶，total RNA，Oligo dT和dNTP。
: cDNA产物用gene specific primer A + RACE primer没有任何条带出来。作为control
: ，用另一个gene specific的primer B和A配对却有条带。说明RT反应没有问题。可是RT
: 反应要进行，RACE primer conjugated oligo dT也要能work才行啊？
: 哪位能说说到底什么地方出问题？ 万分感谢
| B******o
发帖数: 496 | 3 谢谢谢谢。
只是我现在遇到的不是杂带，而是压根没有PCR产物。而gene specific primer则有。
多于3'RACE来说。RT反应的前提是oligo dT能够priming上不是么？为啥会没有条带呢？
3
to
RNAs
same
【在 c********b 的大作中提到】
: I used Invitrogen GeneRACer kit，which suggested nested-pcr.
: Invitrogen's oligo adapter is about 60 nt，first PCR used adapter 5‘-end ~
: 30 nt primer and gene specific（GS) primer. Then they use close to adapter 3
: '-end nested primer and another nested GS primer.
: After the nested PCR, I sometimes still see multiple bands. I have to gel-
: purify the PCR products with correct size and sometimes to repeat 2nd PCR to
: amplify it before cloning.
: One suggestion is to purify mRNA for RACE, which gives you much cleaner
: result. If not, you can try to use LiCl to precipitate large ribosomal RNAs
: before RACE. Also, you can try to use only another GS specific primer+ same
| c********b
发帖数: 363 | 4 Well, I can share some more of my experience with you.
1, Check out the anneal temperature. If no band here, but your positive
control showed no problem for RT, then I would suspect that PCR failed in
your RACE. You can try touch-down PCR or use 2-step PCR (68 degree for
annealing and extension.
2, Increase your PCR cycles. If the annealing temperature is not perfect,
increasing cycle numbers will help. I normally used 36-40 cycles for 2-step
PCR, in some difficult cases. Also, I tried many different Taq enzymes. My
feeling is that Pfu does not show good compatibility to PCR from RT-product
in many cases. I prefer FideliTaq DNA Polymerase from USB. Also, the most
super Taq on my hand for hard cloning is Advantage HD Polymerase from
Clontech. But Advantage HD buffer sucks, which havs to be removed by PCR cleaning kit before subsequent
cloning.
3, oligo dT may produce artifacts by non-specificcally recognizing A-rich
region. I had one experience before, and the 3'RACE only gave me partial
sequence close to 5' end. So you may not be able to rule out the failure of
RT totally.
呢？
【在 B******o 的大作中提到】
: 谢谢谢谢。
: 只是我现在遇到的不是杂带，而是压根没有PCR产物。而gene specific primer则有。
: 多于3'RACE来说。RT反应的前提是oligo dT能够priming上不是么？为啥会没有条带呢？
:
: 3
: to
: RNAs
: same
| f********n
发帖数: 6465 | | S*****s
发帖数: 287 | 6 你也可以重新纯化一下 RNA。我用 Invitrogen 的 3'RACE kit，如果用 5 prime 的
RNA kit 纯化 RNA 就怎么样都做不出来，和你的结果一样什么 PCR 产物都没有。用
Trizol 配上 Ambion 的 RNA kit，同样的 PCR 引物很容易就有 PCR 产物。RNA 的纯
度很重要。 |
|
