McClean: Anneal and Extend

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(New page: <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> ==Overview== This protocol describes a simple procedure to annealing and extending two oligos (with homology) to get double...)
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==Materials==  
==Materials==  
* Oligos (25μM; take the standard McClean Lab -80°C stock which is at 100μM in TE and dilute it 1:4 in H<sub>2</sub>O)
* Oligos (25μM; take the standard McClean Lab -80°C stock which is at 100μM in TE and dilute it 1:4 in H<sub>2</sub>O)
-
* Takara PCR reagents ()
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* Takara PCR reagents (see: [[http://openwetware.org/wiki/McClean:_Takara_PrimeStar_PCR Takara PrimeStar PCR]])
==Protocol==
==Protocol==

Revision as of 16:24, 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

Digestion

The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme β-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely.

  1. Spin down 250 μL of sporulated culture.
  2. Resuspend in 250 μL sterile water.
  3. Mix 17 μL washed culture with 3 μL β-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.
  4. Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time.)
  5. Gently add 100 μL water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.
  6. Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads).
  7. Mark the plate at opposite edges to indicate the center of the plate (see following diagram).
  8. Resuspend the digested cells from your chosen timepoint. by gently tapping or pipetting (they will probably have settled while you were looking at the digestions under the microscope). Drip 20 μl down the imaginary center line you just indicated, beginning from one edge.
  9. Let the digested culture fully absorb into the plate.
  10. Dissect (see dissection section). If you dissect on a different day, you must begin with a fresh plate and set up a new dissection. The sporulation cultures are good for weeks, though the spore viability may start to decrease.

Appearance of Tetrads

Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape.

How to distinguish a tetrad from two budded cells adjacent to each other?

  1. Generally, the spores of a tetrad are smaller than a budded cell.
  2. Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.
  3. The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.


Dissection

Dissecting scopes have a specially designed stage with “clicks” you can feel in both the x and y direction that allow even spacing of tetrads. The micromanipulator is used to control the position of the needle.

  1. Drop the needle to its lowest position by raising the arm of the micromanipulator (yes, this seems somewhat counterintuitive!!)
  2. Move the turret of the scope so that there is no objective lens over the stage. This will give you room to position the plate.
  3. Positioning your agar plate on the stage is like flying a helicopter, not a jet. Landing, come over high, then descend. Taking off, go straight up, then depart. The needle stands higher than the platform!
  4. Position the plate on the stage so that the two marks you made line up with the horizontal axis of the plate.
  5. Swing the objective into position, and move the stage so that the objective is centered over the swath where you dripped the digested sporulation down the plate.
  6. Focus on the cells sitting on the surface of the plate (you will be focusing through the agar into the cells sitting on the surface).
  7. Pick tetrads.
    1. Move the microscope stage so that individual tetrads with a clear zone around them are visible. Raise the needle (by lowering the arm of the micromanipulator) until you can see it.
    2. Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.
    3. Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.
    4. To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.
    5. Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.
    6. Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.
    7. Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.
    8. Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
    9. You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.
    10. Remove the YPD plate from the stage, taking care not to break the microneedle.
  8. Incubate the plate for 2-3 days at room temperature.

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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