s*******s
发帖数: 23 | 1 RT，这边有人做RACE吗？
我的5'RACE一直出不来，都快被老板催死了~
各位有什么经验可以分享一下吗？先谢了！ |
c**********5
发帖数: 653 | 2 我正在做这，PCR 有多个条带， 我都TA 克隆，下周测序 |
W*****o
发帖数: 1780 | |
s*******s
发帖数: 23 | 4 clontech
【在 W*****o 的大作中提到】
: Which Kit?
|
s*******s
发帖数: 23 | 5 5'和3'都出条带了？
【在 c**********5 的大作中提到】
: 我正在做这，PCR 有多个条带， 我都TA 克隆，下周测序
|
c**********5
发帖数: 653 | |
f***n
发帖数: 34 | 7 The kit from Ambion (now ABI) is very good. I did some RACE before. FYI |
C*******e
发帖数: 4348 | 8 只做过3'的RACE,原核,所以要用adaptor
做得是RNA-ligase-mediated-3'-RACE |
s*******s
发帖数: 23 | 9 是吗？
不过老板好像不会让我换Kit
想问一下你们用的什么酶？
是一次PCR就能出条带吗？
我3'的出了，可是5'的一直没出来啊~
【在 f***n 的大作中提到】
: The kit from Ambion (now ABI) is very good. I did some RACE before. FYI
|
f***n
发帖数: 34 | 10 you need to do PCR twice (out primers, and inner primers). I nerver failed
before. |
|
|
s*******s
发帖数: 23 | 11 Thanks! I did it like what you said.
May be I need design some more primers.
【在 f***n 的大作中提到】
: you need to do PCR twice (out primers, and inner primers). I nerver failed
: before.
|
f***n
发帖数: 34 | 12 Yes. You maybe need to redesing primers. Also you need know that 5' RACE is
more challenging. Only for 3' RACE, i bought all the reagents from different
companies and combine to do it and it is cheaper. For 5' RACE, it is very
important to get full length mRNA or cDNA. Some kits can help you to achieve
this. |
f***n
发帖数: 34 | 13 I don't like clontech's RACE kit. Based on my expericne, a lot of cDNA are
not full length. |
x******8
发帖数: 350 | 14 5RACE最重要的是你的RNA质量要好，其实clontech的挺好使的，ambion的比较复杂，我
就没有选。我已经全长克隆了3个基因。 |
f***n
发帖数: 34 | 15 If your expression level is high, you don't have any problem with Clontech's
, but if your expressin level is very low, you will get proble. Also if you
use Clontech's kit to produce cDNA library, you will find that a lot of
clones are not full length, however abion's avoid this problem. This is just
from my experience. |
s*******s
发帖数: 23 | 16 达人啊~
花了多长时间？
【在 x******8 的大作中提到】
: 5RACE最重要的是你的RNA质量要好，其实clontech的挺好使的，ambion的比较复杂，我
: 就没有选。我已经全长克隆了3个基因。
|
s*******s
发帖数: 23 | 17 Thanks!
I want to try abion's if my boss agree to buy a new kit.
's
you
just
【在 f***n 的大作中提到】
: If your expression level is high, you don't have any problem with Clontech's
: , but if your expressin level is very low, you will get proble. Also if you
: use Clontech's kit to produce cDNA library, you will find that a lot of
: clones are not full length, however abion's avoid this problem. This is just
: from my experience.
|
