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Cloning GFP into pCMV-Tag2B vector

GFP IRES promoter Tag2B

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#1 cancerous

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Posted 31 July 2014 - 09:51 AM

Hi all,

 

I am trying to insert GFP into pCMV-Tag2B vector.

 

I found that there is one BstBI site on this vector (one enzyme cloning), right behind NeoR/KanR ORF and I did PCR IRES-GFP region from pMSCV-PIG vector and cloned that PCR product into Tag2B vector to drive GFP expression by the common SV40 promoter, which already drives NeoR/KanR expression.

 

I got a few clones which have the IRES-GFP insert and directly did transfection into 293T cells before sequencing the orientation of the insert.

 

I found that 4 clones showed GFP expression and was very excited. GFP was not so strong compared to GFP in pMSCV-PIG vector but the signal was so obvious. When I sequence them to check the direction, however, all 4 of them were inserted in a reverse direction. How can it be possible? There is no promoter nearby (opposite direction) to drive GFP expression. There is only one promoter in this direction on Tag2B vector (T7 promoter) but it is located very far (2.7kb away from GFP).

 

I did not sequence but I think that rest of the clones (No GFP clones after transfection) may be inserted in a correct orientation, which implies that SV40 common promoter cannot drive GFP expression although there is IRES.

 

Could anyone explain the reason?

 

Or Can I use this vector (I just need GFP+ Tag2B vector!!! and I don't need other vecor. I only need Tag2B for my personal reason!!)?

 

Thanks a lot in advance.


Edited by cancerous, 31 July 2014 - 10:06 AM.


#2 Rajpoot

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Posted 02 August 2014 - 09:11 AM

next time, confirm right orientation of insert before sending recombinant plasmid for sequencing. You can do this by performing colony PCR using forward primer from  plasmid (upstream of Bstb1 site) and reverse primer from insert. 







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