I want to design primer to amplify a gene present on negative strand. My confusion is do I just copy paste that sequence and try to find my primers manually or do I need to get a reverse complement of that sequence to make it (+) strand so I can design my primers?
or I take both strand, design forward primer from (+) strand and reverser primer from (-) negative strand and reverse complement only reverse primer?
I really appreciate any guidance.
"positive" and "negative" strands are just an arbitrary convention. Make sure you are using the coding strand for your gene (e.g. the start codon is 5'ATG . . . ). Use a primer design program like Primer3, don't bother to do this by hand.
The forward/left primer will be an exact match to the coding strand, the reverse/right primer will be the reverse complement. The design program will show you where they anneal, their 5' and 3' directionality, and the exact bases.
Get a colleague to help you. It sounds like you are thinking yourself into circles.
Good luck!
Hi Hira Niaz
Katie is just right, + or - strand doesn't mean anything in PCR since you'll amplify both strands at same time.I don't know for your future purpose but the only important thing is to design right (you can use in silico PCR at UCSC for designing test).
keep safe
fred
I agree with Katie, positive and negative mean nothing in the design
Use one of the primer design programs such as Primer3
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