pEGFP N1 vector - (Dec/10/2007 )
Hi, Dear ALL:
I have one question.
I am building a reporter plasmid now. it has a splice variant form and i want to see it is spliced in the cell. i want to mutate one aa into a stop codon in the splicing site and subclone it into pEGFP N1 vector. SO that if it is not spliced, the GFP will not express ( because of the the stop codon).
my question is, this GFP is in the vector, not attached to my insert and it has its own ATG. so i wonder it will still get expressed even if there is a stop codon ( in the case that it is not spliced). am i wrong?
any suggestion?
i hope i have make myself clear.
Thanks.
I have one question.
I am building a reporter plasmid now. it has a splice variant form and i want to see it is spliced in the cell. i want to mutate one aa into a stop codon in the splicing site and subclone it into pEGFP N1 vector. SO that if it is not spliced, the GFP will not express ( because of the the stop codon).
my question is, this GFP is in the vector, not attached to my insert and it has its own ATG. so i wonder it will still get expressed even if there is a stop codon ( in the case that it is not spliced). am i wrong?
any suggestion?
i hope i have make myself clear.
Thanks.
I guess its doable as long as you have both mature genes aligned in frame. But be sure that splicing may not be a complete event and EGFP is not a very good quantitative reporter.
thank you.
they are in frame; the insert and GFP.
do you mean the GFP is not expressed if there is a stop codon in the insert? there are about 10 nt between the end of instert and GFP.
thanks.
I have one question.
I am building a reporter plasmid now. it has a splice variant form and i want to see it is spliced in the cell. i want to mutate one aa into a stop codon in the splicing site and subclone it into pEGFP N1 vector. SO that if it is not spliced, the GFP will not express ( because of the the stop codon).
my question is, this GFP is in the vector, not attached to my insert and it has its own ATG. so i wonder it will still get expressed even if there is a stop codon ( in the case that it is not spliced). am i wrong?
any suggestion?
i hope i have make myself clear.
Thanks.
I guess its doable as long as you have both mature genes aligned in frame. But be sure that splicing may not be a complete event and EGFP is not a very good quantitative reporter.
Right, but I have seen people putting more than one stop codon just to be sure it really stops.
pEGFP-N1 is a N-terminally tagged vector, right? In your case, wouldn't pEGFP-C series be more suitable? Sorry, I bit confused here.
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no, he/she wants to have the opportunity to stop the expression of egfp through its internal stop codon that can be sliced out.
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It is a C-terminally tagged vector, that is, the GFP will be fused at the C-terminus of the protein, or the protein will be fused at the N-terminus of the GFP, thus cause the name. Confusing, I know.