McClean:Annealing Oligos

Overview

This is a protocol for annealing oligos for yeast transformation.

Materials

• 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

Protocol

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.