McClean:Annealing Oligos

From OpenWetWare
Jump to navigationJump to search


This is a protocol for annealing oligos for yeast transformation.


  • Forward Oligo
  • Reverse Oligo
  • 10X Annealing Buffer
  • 1X TE buffer

Stock Solutions

10X annealing buffer

  • Recipe for 4ml, add
                     400ul 1M Tris pH 8
                     80ul 0.5M EDTA pH8
                     800ul 2.5M NaCl
                     2720ul Water


  1. Resuspend oligos to a stock concentration of 100uM in 1X TE buffer. Store oligo stocks at -20oC when not using.
  2. To a PCR tube, add 5 ul of the proper Top and Bottom strand oligos (reverse complements for each other) and 5 ul of 10X Annealing Buffer and then 35ul water,mix well and briefly spin down . The final concentration of each oligo is now 10 uM.
  3. Incubate the PCR tube in the thermocycler with the program at 95 degree for 3 minutes, then -1 degree every cycle for 60 cycles, at 25 degree for 5 minutes then end.
  4. Use 3ul of the annealed product for yeast transformation.


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.


or instead, discuss this protocol.