McClean:Annealing Oligos: Difference between revisions
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(New page: ==Overview== This is a template for how to write a new protocol in openwetware for our lab. In your real protocol, a description of what the protocol is and when to use it would go here. ...) |
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==Overview== | ==Overview== | ||
This is a | This is a protocol for annealing oligos for yeast transformation. | ||
==Materials== | ==Materials== | ||
* | * Forward Oligo | ||
* | * Reverse Oligo | ||
* | * 10X Annealing Buffer | ||
* | * 1X TE buffer | ||
==Stock Solutions== | ==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== | ==Protocol== | ||
# 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. |
Revision as of 08:05, 24 October 2013
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
- 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.