Smolke:Protocols/Dropout plates

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Current revision (13:18, 28 August 2012) (view source)
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==Notes==
==Notes==
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*Check which kind of YNB you are using. If using the kind that just says w/o Amino acids (rather than w/o Amino Acids and Ammonium Sulfate), use 3.35g of that (rather than 0.85g), and don't add Ammonium Sulfate -Leo
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*Check which kind of YNB you are using. If using the kind that just says w/o Amino acids (rather than w/o Amino Acids and Ammonium Sulfate--which I believe means, and should say, w/o Amino Acids *or* Ammonium Sulfate), use 3.35g/500mL final of that (rather than 0.85g), and don't add Ammonium Sulfate -Leo
*Before autoclaving, the mixture will separate into two layers. It should go into solution after autoclaving.
*Before autoclaving, the mixture will separate into two layers. It should go into solution after autoclaving.
*If pouring multiple bottles of media, the remaining bottles can stay in the autoclave until needed.
*If pouring multiple bottles of media, the remaining bottles can stay in the autoclave until needed.

Current revision

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Contents

Overview

Making selective yeast plates.

Materials

  • Nanopure water
  • Yeast nitrogen base (YNB)
  • Ammonium sulfate
  • Agar
  • Sugars (normally 20% dextrose, alternative 20% raffinose/10% sucrose/20% galactose)
  • Amino acid dropout solution (in 4°C room)

Procedure

N.B. - this protocol makes ~1 sleeve of moderately thick plates. Adjust volumes as needed.

  1. Mix:
    • 400 mL NP water (or less, if using alternate sugars)
    • 0.85g YNB (Stored in cold room)
    • 2.5g NH4SO4
    • 10g Difco agar (not Bacto agar)
  2. Mix the solution well, add a stir bar, and autoclave on liquid cycle
  3. After autoclaving, place flask on stir plate to cool
  4. When bottle is cool enough to touch with the bare hand, add dropout solution and sugar(s)
  5. Mix, then pour into plates
    • Pour just enough cover the base (without shaking)
    • Briefly flame the top of the plate to eliminate bubbles

Notes

  • Check which kind of YNB you are using. If using the kind that just says w/o Amino acids (rather than w/o Amino Acids and Ammonium Sulfate--which I believe means, and should say, w/o Amino Acids *or* Ammonium Sulfate), use 3.35g/500mL final of that (rather than 0.85g), and don't add Ammonium Sulfate -Leo
  • Before autoclaving, the mixture will separate into two layers. It should go into solution after autoclaving.
  • If pouring multiple bottles of media, the remaining bottles can stay in the autoclave until needed.
  • I've found that it cuts down on condensation if you pile the plates on top of each other while they dry. -Josh
  • If you don't want to flame, drop a stirbar into the flask before autoclaving. Mix on the stirplate to add sugar and aa's, stir some more to take care of bubbles. Pour carefully. -Travis
  • An additional bubble-reducing strategy is to use the 70% ethanol spray mist bottle to spray a single mist of ethanol into the flask after mixing in the sugar and aa's but before pouring the plates. Be careful if you flame the flask after spraying, as the ethanol can ignite and send a mini flame shooting out of the flask. -Mike
  • Plate thickness varies greatly depending on who pours the plates. I prefer thicker plates (500mL = 24 plates) rather than thinner plates (500mL = 40+ plates). In my experience, thin plates can dry out before the yeast colonies reach their full growth (so you end up with small colonies and thin streaks). This is particularly problematic for slow-growing strains. -Josh I agree - Mike

Contact

Win

For another protocol, look here.

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