Primer design question - Use of primer 3 (Oct/06/2005 )

Hi everyone.

I read that your primers must be about 30bp upwards and downwards from the target sequence.

I'm using primer3 with the mRNA sequence (1200bp) to design new primers. The target sequence is 550bp (CDs). Should I select 610bp(550+30+30) as target sequence?

What about the product size ranges? Should I choose 500(low) and 610(high)?

My aim is doing TA cloning with this sequence later.

Thank you in advance.

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I've never heard that, and I've never found it necessary.

For example, many times I've used PCR to create a fusion protein (his-tag for example), and my forward primer began with the A of the ATG start codon, while my reverse primer had the gene's stop codon actually in it...

Now, if you want or need to also clone upstream regulatory sequences (the promoter, ribosome binding site, etc.), then you do need to include some bases upstream of the gene; I usually try to take 40 or 50 bases in that case.

I'm working almost exclusively with bacterial genes from chromosomal templates; your mileage may vary if you're working with eukaryotic genes...

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Homebrew already answer the first part of your question, as for the size it doesn't matter between500 or 610bp it will be perfectly fine for TA cloning

things are getting more difficult only if you try to clone a very long pCR product (>5Kb)

pesji

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What you are saying can be true for sequencing primers where it helps to get clean sequencing data if you have a primer atleast 30 bp upstream of the cloned gene in the plasmid for example.

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