McClean: Anneal and Extend
This protocol describes a simple procedure to annealing and extending two oligos (with homology) to get double-stranded DNA.
- 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)
- Dilute oligos to 25μM from the -80°C stock. Dilute into H2O.
- 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
- 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
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 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.
- *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).
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)
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.