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Team:British Columbia/Notebook/Protocols/AnnealedOligonucleotides
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| Line 5: | Line 5: | ||
XbaI, PstI cuts:<br/> | XbaI, PstI cuts:<br/> | ||
| - | <tt>5 CTAGAG------------sequence------------TACTAGTAGCGGCCGCTGCA</tt><br/> | + | <tt>5 CTAGAG-------------sequence-------------TACTAGTAGCGGCCGCTGCA</tt><br/> |
| - | <tt>3 ----TC-reverse complement of sequence-ATGATCATCGCCGGCG----</tt><br/> | + | <tt>3 ----TC--reverse complement of sequence--ATGATCATCGCCGGCG----</tt><br/> |
EcoRI, SpeI:<br/> | EcoRI, SpeI:<br/> | ||
| - | <tt>5 AATTCGCGGCCGCTTCTAGAG------------sequence------------TA----</tt><br/> | + | <tt>5 AATTCGCGGCCGCTTCTAGAG-------------sequence-------------TA----</tt><br/> |
| - | <tt>3 ----GCGCCGGCGAAGATCTC-reverse complement of sequence-ATGATC</tt><br/> | + | <tt>3 ----GCGCCGGCGAAGATCTC--reverse complement of sequence--ATGATC</tt><br/> |
Revision as of 22:36, 22 September 2013
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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 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.
