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[piggybob]

Hello everyone,

I am designing primers for PCR and needed some help with designing them.

I am trying to PCR but by adding about ~40-50 new bps that are not actually present in my template.
It is because Im trying to synthetically link a peptide(~30bp) onto a template encoding a protein with a linker(~15-30bp).
So my final primer design comes to about ~80-90 bp in length.

The problem is that when I run Hairpin/PrimerDimer analysis I get very high deltaG values at like -12kcal/mol.
Also these long primers(Forward or Reverse) have a difference in Tm ~10degrees compared to other primer which is short since it doesnt contain the peptide/linker sequences.

I was wondering how I could get around this problem?!?!

Thanks