3 replies to this topic

[Zhe]

Hi everyone,

I've got a v. basic clonning question here. I've inserted a full-length gene into a pET15b vector. When I got my sequencing results, I found the reading frame of the vector is changed. The plasmid amino acid sequence would be changed upon clonning of my insert in. I'm wondering if this frameshift of the vector dose matter or not?

I'm very new to this, hope someone could help.

Cheers,
Jo

[Denny]

Zhe, on Mar 10 2010, 01:09 PM, said:

Hi everyone,

I've got a v. basic clonning question here. I've inserted a full-length gene into a pET15b vector. When I got my sequencing results, I found the reading frame of the vector is changed. The plasmid amino acid sequence would be changed upon clonning of my insert in. I'm wondering if this frameshift of the vector dose matter or not?

I'm very new to this, hope someone could help.

Cheers,
Jo

This matters very much! If your gene sequence is shifted, and the amino acid sequence will be changed, you won't get the target protein.

Where is the frame shift and what caused it?
Are there extra bases added between the gene and the vector?

[Zhe]

Denny, on Mar 11 2010, 12:50 AM, said:

Zhe, on Mar 10 2010, 01:09 PM, said:

Hi everyone,

I've got a v. basic clonning question here. I've inserted a full-length gene into a pET15b vector. When I got my sequencing results, I found the reading frame of the vector is changed. The plasmid amino acid sequence would be changed upon clonning of my insert in. I'm wondering if this frameshift of the vector dose matter or not?

I'm very new to this, hope someone could help.

Cheers,
Jo

This matters very much! If your gene sequence is shifted, and the amino acid sequence will be changed, you won't get the target protein.

Where is the frame shift and what caused it?
Are there extra bases added between the gene and the vector?

Many thanks 4 ur reply. There is not extra bases added between the gene and vector. Actually, I used the same primers as clonning of the same gene into a similar vector pET3a. The sequencing results for that clonning was correct.
Do u hv any idea of wat could possibly cause the frameshift plz?

[Denny]

Zhe, on Mar 11 2010, 10:53 AM, said:

Denny, on Mar 11 2010, 12:50 AM, said:

Zhe, on Mar 10 2010, 01:09 PM, said:

Hi everyone,

I've got a v. basic clonning question here. I've inserted a full-length gene into a pET15b vector. When I got my sequencing results, I found the reading frame of the vector is changed. The plasmid amino acid sequence would be changed upon clonning of my insert in. I'm wondering if this frameshift of the vector dose matter or not?

I'm very new to this, hope someone could help.

Cheers,
Jo

This matters very much! If your gene sequence is shifted, and the amino acid sequence will be changed, you won't get the target protein.

Where is the frame shift and what caused it?
Are there extra bases added between the gene and the vector?

Many thanks 4 ur reply. There is not extra bases added between the gene and vector. Actually, I used the same primers as clonning of the same gene into a similar vector pET3a. The sequencing results for that clonning was correct.
Do u hv any idea of wat could possibly cause the frameshift plz?

Unless you've done PCR on the vector to amplify the whole vector, I don't know how a base could have been inserted or deleted outside of the cloning site and restriction sites of the vector.

Did you use the same restriction enzymes for 15b & 3b ?

Maybe a lab member could take a look at your protocol, sequencing results, compare it to the vector & gene reference sequences and put some fresh eyes on the problem.
Sorry not much help.

Edited by Denny, 11 March 2010 - 05:59 PM.