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Stop codon problem
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| oioi |
Posted on 09-04-2010 02:33
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Member
Posts: 2
Joined: 09.04.10
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I have have run a pilot experiment in vivo for a gene X, delivered in plasmid pcDNA3.1/V5-HisŠTOPOŽTA plasmid from invitrogen with marginal/none results. Careful sequenching of the plasmid+gene insert after experiments showed that somehow my stopcodon has been altered/edited/mutated so that it reads TAAG instead of TAG. Have any of you an ide of how much less, if any this will effect my expression and my experiments...? Could it explain why my experiments were not successful?
regards
a novice |
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| AP |
Posted on 11-04-2010 21:25
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Member
Posts: 4
Joined: 15.03.10
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This is not likely the cause of your problem(s). Translation should have stopped at TAA, because TAA is also a stop codon; along with TAG and TGA. |
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| poley |
Posted on 11-04-2010 21:45
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Super Administrator
Posts: 1
Joined: 22.02.10
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According to an article in the Journal of Molecular Evolution from September of 2005; The stop codon may have an effect on the expression levels.
"UAA is overrepresented in the lower eukaryotes, UGA is overrepresented in the higher eukaryotes."
http://www.springerlink.com/content/rq5433859ltwp2g0/ |
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| DNADisaster |
Posted on 14-04-2010 02:55
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Member
Posts: 2
Joined: 14.04.10
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Are you able to test the DNA as a vaccine to elicit CTL response? I have a vaccine that I cant detect protein expression from in mammalian tissue, but it gives a great CL response to encoded epitopes... |
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| oioi |
Posted on 16-04-2010 08:08
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Member
Posts: 2
Joined: 09.04.10
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Thanks for good suggestions, guys. I was thinking perhaps to do a cell-line transfection experiment, to see if I can get translation of the protein.. It would perhaps quickly give me an answer if there is expression from the plasmid or if the construct fails to do so... |
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