RobWardenWNB:M3D3

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Expression Engineering, Day 3, 4/11/07

Counting Colonies

Transformation

  • No DNA: 0
  • + DNA: 0
  • pRS416: 94 x 8 = 752

Result Interpretation: Our positive and negative controls verify the transformation. The much lower count of positive colonies than the other groups is consistent with using the plated yeast as opposed to those in log phase growth. Perhaps if the yeast were in log phase we would have had useful transformants, however we will now need to use another strain to continue our experiment.

Medias

  • -ura ON: 0
  • -trp ON: 149
  • YPD ON: 147
  • -ura log: 0
  • -trp log: 102 x 8 = 816
  • YPD log: 97 x 8 = 776

Result Interpretation: The cells cannot produce their own uracil. They are just as viable with or without Trp, because they can make their own.

New Strain

NY389: MATa ura3-52 his4-917delta leu2d1 trp1-63 spt8D-302::LEU2
See: Martens, Wu, Winston, Genes & Development

Due to the lack of transformants with our PCR construct, we will be using a new strain of yeast which already contains an SPT8 knockout. The new reporter is LEU2.

Part 1: Colony PCR

Purpose
To make sure our new strain is what we expect it to be.

Procedure:

  1. On the tip of a sterile toothpick, pick-up cells from the plate and swirl the cells in 20 μl of sterile water in an eppendorf tube. Repeat two more times.
  2. The teaching faculty will have the parent strain, FY2068, streaked out on a petri dish. You should mix 1/2 of a colony from this plate with 20 μl of sterile water in a fourth eppendorf tube.
  3. Close the caps to the tubes and microwave them in an eppendorf rack for 15 seconds.
  4. Move 2.5 μl of the microwaved mixes, yeast debris and all, into a 200 μl PCR tube. Again label your tubes well (write small!).
  5. In a new eppendorf tube (normal size this time), prepare a PCR cocktail enough for 5 reactions. Each reaction will include:
  6. Add 22.5 μl of PCR cocktail to each PCR tube with yeast and leave the tubes on ice until everyone is ready.
  7. Cycle the reactions as:
    • 95° 4 minutes
    • 95° 1 minute
    • 52° 1 minute
    • 72° 2 minute
    • repeat steps 2-4 35 times
    • 72° 10 minutes
    • 4° forever

Reagents
PCR Cocktail:

10 μL   x5 = 50 μL   2.5X PCR mastermix
0.5 μL  x5 = 2.5 μL  forward primer specific for SPT8.
0.5 μL  x5 = 2.5 μL  reverse primer specific for LEU2.
11.5 μL x5 = 57.5 μL sterile water

Overnight cultures

Purpose
To prepare yeast for expression analysis on Friday

Protocol:

  1. Using sterile technique, aliquot 2.5 ml of YPD into four sterile test tubes.
  2. Label the caps with your team color, and either "parent" "A" "B" or "C."
  3. Use sterile dowels to move the second 1/2 of each yeast colony from your transformation plates to the media. Swirl the dowel to remove the yeast from the stick and vortex the solution to fully resuspend them.
  4. Use a sterile dowel to innoculate the "parent" culture, using the FY2068 plate that the teaching faculty have prepared.
  5. Place the tubes on the roller drum in the 30° incubator. They will grow for 24 hours and then be placed in the 4° fridge until next lab.

Summary

Today was a day of preparation and validation. The PCR products will verify that our knock in is where we want it to be. The overnight cultures are preparing the yeast cells to be used for further experimentation on Friday.