Skip to main content
1 of 4

Designing primers with restriction sites

I want to design a forward and reverse primer that include overhangs with restriction sites.

If I want to send my dna to a synthesis company should I include the attached overhang with the restriction sites to the dna? Does it matter if the primers have the overhang of the dna within their sequence ?

From the sequence right at the end what bp sequence would be recommended I use for my primers if I want 20bp for the primer?

See below for cDNA sequence:

Original cDNA sequence

5' ATGGGTTCAAACACTTCCAAAGTGGGTGCTGGTGCAGAAAAACAACAAGTCTATACTCCG
CTAACACAGATCGATTTTTCACAGTCTTTGGTTTCTCAATTGGATTCATCGAAGGAATCA
GACTATGTCACCAAGCAAAATGCAGAAAAGTTCATTGAGAAGAAGGTTTCACAAAGGCTA
TCTAACCTAGAAGTTGAAACGTTAAAGAAGTTTGAAGATACTTTGAACAATTCACTATTA
TCAGACGACGACAAGGATGCCGTTGATGGAATATCATCAAGTTCATTGAATAATCAAATC
GAGTCGTTGAACAAGAAACTAACATTATTTGATCAATTAGAGTTACAAAAGTTGGAGAAA
TATGGGGGTGCCAAAGGTAAATCTGATAAAAAAACCGACAACGGCAGCATTTCTATAAAG
GCAAAATTGACTGAGTGTCTTTTGGCCAATAAGGGCAAGCCATTGAATTGTTACGAAGAG
ATGGAAGAATTCAAGAAGCTCGTTATGGGTTGA 3'

Nde1 cut site: 5' ... CA|TATG ... '3'

Xho1 cut site: 3'...GAGCT|C...5'

cDNA with overhangs that have restriction sites, Ndel1 (5'->3') and Xho1 (3'->5')

5' catatg - ATGGGTTCAAACACTTCCAAAGTGGGTGCTGGTGCAGAAAAACAACAAGTCTATACTCCG
CTAACACAGATCGATTTTTCACAGTCTTTGGTTTCTCAATTGGATTCATCGAAGGAATCA
GACTATGTCACCAAGCAAAATGCAGAAAAGTTCATTGAGAAGAAGGTTTCACAAAGGCTA
TCTAACCTAGAAGTTGAAACGTTAAAGAAGTTTGAAGATACTTTGAACAATTCACTATTA
TCAGACGACGACAAGGATGCCGTTGATGGAATATCATCAAGTTCATTGAATAATCAAATC
GAGTCGTTGAACAAGAAACTAACATTATTTGATCAATTAGAGTTACAAAAGTTGGAGAAA
TATGGGGGTGCCAAAGGTAAATCTGATAAAAAAACCGACAACGGCAGCATTTCTATAAAG
GCAAAATTGACTGAGTGTCTTTTGGCCAATAAGGGCAAGCCATTGAATTGTTACGAAGAG
ATGGAAGAATTCAAGAAGCTCGTTATGGGTTGA - gagctc 3'