McClean:Annealing Oligos

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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==
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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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This is a protocol for annealing oligos for yeast transformation.
==Materials==  
==Materials==  
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* Item 1
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* Forward Oligo
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* Item 2
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* Reverse Oligo
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* Stock Solution 1
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* 10X Annealing Buffer
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* Stock Solution 2
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* 1X TE buffer
==Stock Solutions==
==Stock Solutions==
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'''Stock Solution 1'''
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'''10X annealing buffer'''
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* This is a very simple solution, so we only need a one line description of how to make it.  
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* Recipe for 4ml, add
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                      400ul 1M Tris pH 8
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                      80ul 0.5M EDTA pH8
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                      800ul 2.5M NaCl
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                      2720ul Water
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'''Stock Solution 2'''
 
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This is a more involved solution, so we will describe how to make it in several steps:
 
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# Step 1
 
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# Step 2
 
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# Step 3
 
==Protocol==
==Protocol==
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# Resuspend oligos to a stock concentration of 100uM in 1X TE buffer. Store oligo stocks at -20oC when not using.
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# 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.
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# 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.
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# Use 3ul of the annealed product for yeast transformation.

Revision as of 11:05, 24 October 2013

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.
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