wolfsc at ibms.sinica.edu.tw
Wed Jan 30 04:41:21 EST 2002
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
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
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.o.C.
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