McClean:Annealing Oligos

From OpenWetWare

Revision as of 10:05, 24 October 2013 by Ping Xu (Talk | contribs)
Jump to: navigation, search

Contents

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.
Personal tools