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Team:British Columbia/Notebook/Protocols/AnnealedOligonucleotides
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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: | 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 | + | ====XbaI, PstI==== |
| - | <tt>5 CTAGAG-------------sequence-------------TACTAGTAGCGGCCGCTGCA</tt><br/> | + | <tt>5' CTAGAG-------------sequence-------------TACTAGTAGCGGCCGCTGCA</tt><br/> |
| - | <tt>3 ----TC--reverse complement of sequence--ATGATCATCGCCGGCG---- | + | <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=== | ===Phosphorylate Oligonucleotides=== | ||
| - | If the cut vector has been dephosphorylated, either order 5’ phosphorylated oligonucleotides or phosphorylate the 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=== | ===Anneal Oligonucleotides=== | ||
Latest revision as of 01:12, 23 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
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.
