McClean: Anneal and Extend

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(Protocol)
(Protocol)
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#**53°C for 20s  
#**53°C for 20s  
#**72°C for 30s
#**72°C for 30s
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#*72°C 5 min
#*72°C 5 min

Revision as of 17:44, 19 September 2012


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. Dilute oligos to 25μM from the -80°C stock. Dilute into H2O.
  2. 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
  3. Run in a thermocycler as follows (notice that we do not use many cycles):
    • 94°C for 5 min
    • Repeat the following three (3) 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.

  1. *Megan N McClean When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit. It is absolutely infuriating, if not impossible, to try dissection on plates that are wet.
  2. *Megan N McClean Different tetrad dissection protocols call for using different enzymes to digest the ascus wall. Our protocol uses a β-glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail). Zymolyase, another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at the β-1,3-glucan linkages releasing laminaripentaose as the principal product. β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans).

References

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

or instead, discuss this protocol.

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