Help!! transcription of Antisense strand

[Peter Ashby]

In article <20020718040315.25047.qmail at ww02.hostica.com>,
 kunika_jain at rediffmail.com wrote:

> 
> 
> Dear All ,
> 
> I have a conceptual problem. I want to produce a PCR  template for 
> transcription of Antisense strand.
> 
> Please verify if my understanding is right, if I say:
> 
> 1. The promoter sequence will be added to the 5' end of the downstream or 
> antisense primer.  If yes then what will be the status of the stop codon that 
> is present as the very last codon of my ORF?? 

it will no longer be the stop codon since you are transcriping the 
antisense, or non coding strand.

> 2. What will be the significance of the added tag that comes after the stop 
> codon.

Since it usually forms only  a very small percentage of the probe, very 
little.
 
> 2. Is it the right approach?

Yes, though I have found I get better efficiency with some sequence 
5prime of the polymerase promoter.
 
> 3. In this case do I need to have a start codon in my promoter tag it self.

No. I assume you want to make antisense for a probe. In this case start 
and stop codons are meaningless since you are only making rna not 
translating it into protein.

Peter

-- 
Peter Ashby
Wellcome Trust Biocentre
University of Dundee, Scotland
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