Designing Primers - (Oct/09/2008 )

Hi I am trying to design primers from a sequence does my method make sense for my 5' primer:

5' (4-6 base pairs that are GC rich) - (RE) - (ATG) - (6 amino acids)

Making sure that there is more than 40% GC content

I was told to add 4-6 base pairs at the 5' end and I was wondering why that is?

Thanks in advance.

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4-6 bp is for restriction enzyme to Dock on the sequence efficiently and cleave the RE site. Without the extra base overhang, restriction digest might not proceed as the RE can't bind to that site strongly and "slide off" before being able to cut.

As for the ATG thing . . As long as the sequence is IN-frame , you're all set. looks like u 're gonna do protein expression study from the primer design.

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