Team:British Columbia/Notebook/Protocols/AnnealedOligonucleotides

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2014 UBC iGEM

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


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 enzymatically phosphorylate the oligonucleotides.

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.