This is a protocol for annealing oligos for yeast transformation.
- Forward Oligo
- Reverse Oligo
- 10X Annealing Buffer
- 1X TE buffer
10X annealing buffer
- Recipe for 4ml, add
400ul 1M Tris pH 8 80ul 0.5M EDTA pH8 800ul 2.5M NaCl 2720ul Water
- Resuspend oligos to a stock concentration of 100uM in 1X TE buffer. Store oligo stocks at -20oC when not using.
- 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.
- 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.
- 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!
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- Ping Xu 24 October 2013 (EDT)
or instead, discuss this protocol.