2 replies to this topic

[chrisbelle]

I have an ORF that I want to clone into the pBAD topo-ta expression system. What do they mean when they say (in the protocol) that the primer design should be:

1. - to include the V5 epitope and polyhistidine region, I should design the reverse PCR primer to remove the native stop codon in the gene of interest and preserve the reading frame through the C-terminal tag.

2. - not include the V5 epitope and polyhistidine region, I should include the native seq containing the stop codon in the reverse primer or make sure the stop codon is upstream from the reverse PCR primer binding site.

Does (1.) mean the reverse primer should include the stop codon region but not the stop codon sequence? Or design the reverse primer right before the stop codon? If so wouldn't the peptide sequence be incomplete?

Please help, I have been doing genomics all along, this is my first touch with proteomics...

Thanks, people...
Chris

[theprawn]

chrisbelle, on Apr 6 2009, 03:36 AM, said:

I have an ORF that I want to clone into the pBAD topo-ta expression system. What do they mean when they say (in the protocol) that the primer design should be:

1. - to include the V5 epitope and polyhistidine region, I should design the reverse PCR primer to remove the native stop codon in the gene of interest and preserve the reading frame through the C-terminal tag.

2. - not include the V5 epitope and polyhistidine region, I should include the native seq containing the stop codon in the reverse primer or make sure the stop codon is upstream from the reverse PCR primer binding site.

Does (1.) mean the reverse primer should include the stop codon region but not the stop codon sequence? Or design the reverse primer right before the stop codon? If so wouldn't the peptide sequence be incomplete?

It depends on your application.  If you are purifying using the 6xHis tag ('polyhistidine region'), which is a very common method, then your reverse primer should NOT include the stop codon.

ex.
          ... AAG AAG TGA     <--your ORF's 3' end
          ...  TTC TTC             <--your reverse primer


This will create a fusion protein with your ORF and the V5 (recognized by anti-V5 antibodies to quantify expression) and 6xHis tags.
If you do not want a fusion protein, then include that stop codon.


As I understand it, the stop codon is not considered part of the protein, so omitting it still produces a 'complete' polypeptide.

[chrisbelle]

THanks!!!