Team:British Columbia/Notebook/Protocols/AnnealedOligonucleotides

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==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====
<tt>5' CTAGAG-------------sequence-------------TACTAGTAGCGGCCGCTGCA</tt><br/>
<tt>3' ----TC--reverse complement of sequence--ATGATCATCGCCGGCG----</tt><br/>

====EcoRI, SpeI====
<tt>5' AATTCGCGGCCGCTTCTAGAG-------------sequence-------------TA----</tt><br/>
<tt>3' ----GCGCCGGCGAAGATCTC--reverse complement of sequence--ATGATC</tt><br/>

===Phosphorylate Oligonucleotides===
If the cut vector has been dephosphorylated, either order 5’ phosphorylated oligonucleotides or [[Team:British_Columbia/Notebook/Protocols/T4PNK|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.