Team:Hawaii/Protocols/oligo hybridization

From 2008.igem.org

Hybridization of Parts

Protocol

  1. Mix:
    3 μl 100 µM sense oligo
    3 μl 100 µM anti-sense oligo
    3 μl 10 x PNK (polynucleotide kinase) buffer
    2 μl 10mM ATP
    2 μl T4 polynucleotide kinase (PNK)
    17 μl distilled water
    Total volume = 30 μl
  2. Incubate at 37C for 1.5 hours.
  3. Add 4 μl 0.5 M NaCl.
  4. Heated mixture in PCR machine to 97C for 2 min. Cooled mixture at each degree between 80 and 96 for 2 min (Tm range of oligos). Cooled mixture at each degree between 25 and 79 for 1 min.
Reference: Pam Silver Lab. [http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts| Oligonucleotide Inserts.]