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 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.
This is a protocol for annealing oligos for yeast transformation.


==Materials==  
==Materials==  
* Item 1
* Forward Oligo
* Item 2
* Reverse Oligo
* Stock Solution 1
* 10X Annealing Buffer
* Stock Solution 2
* 1X TE buffer




==Stock Solutions==
==Stock Solutions==


'''Stock Solution 1'''
'''10X annealing buffer'''
* This is a very simple solution, so we only need a one line description of how to make it.  
* Recipe for 4ml, add
                      400ul 1M Tris pH 8
                      80ul 0.5M EDTA pH8
                      800ul 2.5M NaCl
                      2720ul Water


'''Stock Solution 2'''
This is a more involved solution, so we will describe how to make it in several steps:
# Step 1
# Step 2
# Step 3


==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

  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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