Is it total ORF is required for complete gene expression - (Jun/04/2008 )

Hi,
I want to overexpress one gene, for that i shulod clone whole ORF or part of ORF is sufficient to overexpress under the Hsp promoter.
another question if put total orf region for primer designing to isolate 1116bp in software programme i am getting product size of this primer is only 200 to 300 bp.if it is like this how can i isolate total orf of this gene to express completely.please help to isolate total 1116bp orf.
Thankingyou very much.
SVRREDDY
Ph.D student.
Italy

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First question depends on what next step you will do, what purpose is your experiment. Second one you can design primers for ORF by hand to caluclate Tm using idtdna online oligoanalyzer, or you can amplify several fragments for further assembly by ligation to get full ORF. Good luck

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SV, from your previous question and this, I think you are terribly behind in your reading. Not ridiculing, but you do need to read a lot, if you have not already started.

You need entire ORF to overexpress entire protein. If you have anything less than ORF,

(a) protein is not full length, truncated --which may have a completely different or no function.
(b) such protein may not be expressed at all (if start codon is missing),
© may not be stable (if any part is missing).
(d) may be out of frame, encoding a garbage and truncated sequence.

Please let me know you are planning to read basic molecular biology.

..

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the orf is not enough.
To overexpress in E.coli you need a promoter (ex t7, lac, etc), a shine delgano sequence, etc. These elements are readily available in most expression system. Often times, it's an inducible system with lacO and IPTG to induce the expression.

To overexpress in mammalian cells, you need more. You need to decide if you want transient expression or stable transfection. If stable you need to select and clone out the cells. You'll need a promoter and a polyA signal in the very least. Sometimes, you need a 5'UTR and splice donor/acceptor for efficient mRNA processing to occur. Alot of things to consider....

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