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about sequencing pCMV

about sequencing pCMV# Biology - 生物学

h*g2014-07-24 07:07

1 楼

I bought a pCMVsport6 vector with one gene inserted. I want to confirm the
sequence of the gene. Using T7 primers, I can get part of the sequence. But
using sp6, M13 forward and reverse primers, I cannot get any sequence. Why?
Can anyone help me about this?
Thanks.

l*a2014-07-24 07:07

2 楼

不能用cmv protomoter 来sequence？

But
?

【在 h*********g 的大作中提到】

: I bought a pCMVsport6 vector with one gene inserted. I want to confirm the
: sequence of the gene. Using T7 primers, I can get part of the sequence. But
: using sp6, M13 forward and reverse primers, I cannot get any sequence. Why?
: Can anyone help me about this?
: Thanks.

l*y2014-07-24 07:07

3 楼

cmv的sequence google一下就有了，很多测序公司都有cmv的引物
实在不行用你gene的sequence去搞几个primer测几段overlap的自己拼

But
?

【在 h*********g 的大作中提到】

h*g2014-07-24 07:07

4 楼

I have not tried cmv promoter. But sp6 or M13 reverse is more closer to the
3' end of my gene. Any suggestions? Thanks.

【在 l******a 的大作中提到】

: 不能用cmv protomoter 来sequence？
:
: But
: ?

h*g2014-07-24 07:07

5 楼

Next I will use restriction enzyme to cut, and use T7 and SP6 to make probes
. So I want to know the correct sequence. Why sp6, M13forward and reverse
not working? Thanks.

But
?

【在 h*********g 的大作中提到】

T*e2014-07-24 07:07

6 楼

不知道有个小细节楼主注意过没有，有的时候通用引物的序列可能是反的
不过你说完全没有测出来，是引物有问题吗？
如果你有那个插入的gene序列的话，我建议你自己每隔500bp设计一个引物，把这个基
因测通

f*f2014-07-24 07:07

7 楼

买的载体总会有序列，公司的通用引物也该有序列吧。比对一下看看具体在哪有没问题。
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是指sequence of insertion还是完全没信号，假如引物跟载体能匹配的话估计就是测
序的问题了，想测哪段自己合成引物也成。