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Poor expression in my cloned vector (pMSCV-PIG)

pMSCV-PIG

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#1 cancerous

cancerous

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Posted 05 March 2013 - 08:10 AM

Dear experts,

I have cloned my gene of interest into pMSCV-PIG vector. The brief insert sequence is as followed;

CTCGAG(= XhoI) GCCACC (=Kozak) ATG-------TAA (ORF) GTTAAC (=HpaI)

I used two enzyme sites for this cloning (XhoI and HpaI). In transient transfection in 293T cells, I could not get any overexpression.

I haven't used pMSCV-PIG vector before, so don't know if inserting ORF without extra mRNA sequence at 5' and 3' ends is enough.

Could anyone tell me any suggestion?

Thanks.

Cancerous

#2 bob1

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Posted 05 March 2013 - 11:40 AM

It should be fine to insert without any UTRs.

What times have you tried to detect the protein at following transfection? Have you looked to see if the mRNA is expressed?




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