McClean:Annealing Oligos: Difference between revisions
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==Contact== | ==Contact== | ||
<!--Change the information below to your info if you add a new protocol--> | <!--Change the information below to your info if you add a new protocol--> | ||
*'''[[User:Ping Xu|Ping Xu]] | *'''[[User:Ping Xu|Ping Xu]] 24 October 2013 (EDT)''' | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
Latest revision as of 08:52, 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.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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.
Contact
- Ping Xu 24 October 2013 (EDT)
or instead, discuss this protocol.
