"

From 2013.igem.org

iGEM Home

Contents 1 Assembling short parts from annealed oligonucleotides 1.1 Oligo design 1.2 Phosphorylate Oligonucleotides 1.3 Anneal Oligonucleotides 1.4 Ligate oligonucleotides

Assembling short parts from annealed oligonucleotides

Oligo design

Add the following prefix and suffix to both the sense and antisense sequences, depending on which enzymes you are planning on cutting your vector with:

XbaI, PstI cuts:
5 CTAGAG-------------sequence-------------TACTAGTAGCGGCCGCTGCA
3 ----TC--reverse complement of sequence--ATGATCATCGCCGGCG----

EcoRI, SpeI:
5 AATTCGCGGCCGCTTCTAGAG-------------sequence-------------TA----
3 ----GCGCCGGCGAAGATCTC--reverse complement of sequence--ATGATC

Phosphorylate Oligonucleotides

If the cut vector has been dephosphorylated, either order 5’ phosphorylated oligonucleotides or phosphorylate the oligonucleotides (e.g. with T4 PNK).

Anneal Oligonucleotides

Mix sense and antisense sequences at equimolar concentrations in TE pH 8.0 buffer. Heat to 95 °C for 3 minutes and slowly cool to 25 °C at 2 °C per minute.

Ligate oligonucleotides

Add 60-70 ng of the annealed oligonucleotides to 10-15 ng of cut, dephosphorylated vector. Ligate and transform according to the standard protocol.