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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<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
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 <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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 <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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*[[User:Megan N McClean|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.
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*[[User:Megan N McClean|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==
==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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