Primer doubts - (Aug/25/2008 )

how much is the ideal GC content in primers?

Also when does one do 5' phosphorylation of primers?

Is there any software available online for me to do an in silico PCR of a plasmid [in my case pcDNA3] with the insert of m interest?

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gc content=50%

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The melting temperature ™ of your primer, lack of hairpin loop structure and primer-primer hybridisation is often the more important factor than GC content.

5' phosphorylation of primers is used when you want to clone your insert via a blunt end-blunt end ligation to the vector. It is sometimes used when the sequence of your insert is unknown, or has too many restriction sites.

However the enzyme PNK can add 5' phosphate onto your insert. And since PNK is cheap (last I look) and 5'phosphorylated cost money. Some labs do not buy 5'phosphorylated primers and used PNK instead.

Also note, TA vectors are commonly used when ligating inserts of unknown sequence. The extra A overhang on the insert (Added by Taq, or treatment with Taq after the PCR), makes the ligation much more easier.

I would also not recommend doing a blunt end-blunt end ligation as they aren't as easy to do compared to sticky end ligation. Adding your desired restriction sites to the 5' end of your primer is an easy method to ligate your insert into your vector.

I am sorry, I can't help with the last question. I am sure somebody else in the forum can answer that.

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