Sequencing primers

Wolfgang Schechinger wolfsc at
Wed Jan 30 04:41:21 EST 2002

Dear Kunika,

I assume you will use some automated dye terminator sequencing

I that case you got it! Have the distance between A1 and B1 at least 200
not more than 899 :) bases to make sure you get good overlapping sequence

For closer considerations, estimate that you may be able to get data at a
worse of 100 bp downstream of primers and that you may *clearly* read 500
600 bases. The primer design itself is not so critical when you will use
plasmid. You might test them in a "normal" PCR reaction. Tm should be
55 degC.

Regards and good luck, 


> Dear All,
> I wish to design custom primers for sequencing. All the sequencing primer
> design guidlines I read states that the primers should Generally,be design in
> the region where you have confidence in the accuracy of the sequence from which
> the primer is drawn. Primers on opposite strands should be placed in staggered
> fashion as much as possible. (1) So am I rigth if a choose to design all my
> forward primers at an interval of 350 -500 bases? (2) If I want to stagger the
> primer on opposite strand what should be the distance between the forward and
> reverse primer?
>  A1		    A2			A3		A4
> ------>           ------->          ------->        
> _______________________________________________________________________ 
>        <-------            <------           <------
>   B1		  B2		   B3
> (3) Does the above diagram describes what is meant by stagerring? 
> (4) What should be the distance between A1 and B1? 
> Have I anywhere misinterpreted the design guidelines. 
> Any comment will a great help. Please excuse if I am being dense.
> Thank you 
> Sincerely,
> Kunika
> <>

Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.o.C.


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