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[Sticks]

General problem: I don't know if the output primer sequences are both 5'- to 3' or if only the left primers are given in that format?

Ok now the other issues.

I obtained the sequence from NCBI. It gives the sequence, 3' to 5'. Does this make a difference when I input it. Should I reverse the sequence myself before I input it?

Lets start with this for now.

Any help is greatly appreciated

[methylnick]

Hi sticks,

to answer your question, all primer design programs will give you the primer sequence in 5'-3' orientation. This is the same for methprimer, when designing primers it's always handy to check the primer sequence against the template you were designing from as a double check.

As for NCBI sequence if you are given the 3'-5' sequence then that is the sequence that is used for primer design. Remember that methylation work is strand specific and this doesn't really matter so much in mamalian systems. It's better to reverse complement your NCBI sequence first.

Nick