McClean: Anneal and Extend

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==Protocol==
==Protocol==
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# Design oligos so that they have ~20bp of overlap.
# Dilute oligos to 25μM from the -80°C stock.  Dilute into H<sub>2</sub>O.
# Dilute oligos to 25μM from the -80°C stock.  Dilute into H<sub>2</sub>O.
# For one reaction the PCR mix should be:
# For one reaction the PCR mix should be:
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==References==
==References==
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Adapted from Maitreya Dunham's protocol (http://dunham.gs.washington.edu/sporulationdissection.htm) and the Botstein lab protocol (http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf)
 
==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-->
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*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''
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*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 19 September 2012 (EDT)'''
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Current revision


Contents

Overview

This protocol describes a simple procedure to annealing and extending two oligos (with homology) to get double-stranded DNA.

Materials

  • Oligos (25μM; take the standard McClean Lab -80°C stock which is at 100μM in TE and dilute it 1:4 in H2O)
  • Takara PCR reagents (see: Takara PrimeStar PCR)

Protocol

  1. Design oligos so that they have ~20bp of overlap.
  2. Dilute oligos to 25μM from the -80°C stock. Dilute into H2O.
  3. For one reaction the PCR mix should be:
    • 10μL 5X Takara Buffer
    • 4μL 2.5mM DNTPs
    • 0.5μL Takara polymerase
    • 1 μL 25μM forward oligo
    • 1 μL 25μM reverse oligo
    • 33.5μL H2O
  4. Run in a thermocycler as follows (notice that we do not use many cycles):
    • 94°C for 5 min
    • Repeat the following three lines 2-3 times:
      • 98°C for 10s
      • 53°C for 20s
      • 72°C for 30s
    • 72°C 5 min

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  • Megan N McClean It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well.

References

Contact

or instead, discuss this protocol.

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