20.109(S21):M1D3

Introduction:Harvest candidate clones

Protocols:

The yeast that were sorted via FACS in our last lab session were incubated at 30°C for an additional three days so the yeast could recover from the sorting and grow. This additional growth time will give us a sufficient number of cells to harvest plasmid DNA.

Part 1: Harvest plasmid DNA from yeast

To harvest plasmid DNA from yeast we need to do a bit more work to break down the cell wall. Yesterday the 2x10⁷ yeast cells were pelleted and digested with zymolase overnight at 37°C. Zymolase is a mixture of enzymes purified from the bacteria Arthrobacter luteus that degrades the cell wall of yeast and other fungi. The yeast plasmid purification will be carried out using reagents from a commercial kit called Zymoprep MiniPrep II.

1. Retrieve your overnight yeast digests from the 37°C incubator.
2. Add 200uL of Solution 2 and vortex briefly.
   • This is the alkaline buffer.
3. Add 400uL of Solution 3, vortex briefly and centrifuge at 14,000g for 10min.
   • This is the buffer that neutralizes the pH.
4. Transfer the supernatant to a new 1.5 mL tube and centrifuge at 14,000g for an additional 10min.
5. Transfer the supernatant to a blue DNA binding, silica column and centrifuge at 10,000g for 30sec. Discard the flow through into a tube labeled 'zymo waste'.
6. Add 550uL DNA wash buffer to the column, centrifuge at 10,000g for 2min, and discard the flow through to 'zymo waste'.
7. Transfer the column to a new 1.5 mL tube and carefully add 10uL of water to the center of the column to elute the DNA.
8. Let the column sit for 2min at room temp.
9. Centrifuge at 10,000g for 1min to elute the plasmid DNA from the column and collect the flow-through.
10. You will now take your samples to the Nanodrop to measure the DNA's absorbance to quantify the concentration.

Part 2: Transform plasmid from yeast into E. coli

Competent cells are fragile and should be handled gently, specifically kept cold and not vortexed.

1. Label a 1.5 mL tubes with your team information and chill it in your ice bucket.
2. Carefully aliquot 25uL of competent NEB 5-alpha E. coli cells from the front laboratory bench in to your cold tube on ice.
3. Add 5uL of the yeast plasmid DNA to the competent bacteria.
   • Gently tap the tube with your fingertip 5 times to mix.
   • Remember: it is important to keep the competent cells cold. Also, avoid over pipetting and vortexing!
4. Incubate your transformation mix on ice for 30 min.
5. Carry your ice bucket with your transformation to the heat block at the front laboratory bench.
   • Be sure you also take your timer.
6. Transfer the tube with your transformation to the heat block set to 42 °C and incubate for exactly 30 sec.
7. Remove your transformation from the heat block and immediately put them back in the ice bucket, then incubate for 5 min.
8. Pipet 500 uL of pre-warmed SOC media into the transformation.
9. Move your transformation to the 37 °C incubator and carefully place them on the nutator.
10. Incubate transformation for 1 h.
11. Retrieve your transformation from the incubator and alert the teaching faculty that you are ready to plate your samples.
12. Plate 100μL of the transformation onto an appropriately labeled LB+Amp agar plate.
    • The teaching faculty will demonstrate how you should spread the transformation onto the LB+Amp agar plate by using an ethanol burner to sterilize a glass cell spreader.
13. Move your spread plate to the 37 °C incubator where they will incubate overnight.

Bacterial transformation: Blue cells did not take up plasmid during heat shock and red cells did take up plasmid with antibiotic resistance marker during heatshock.

Reagents

• Zymoprep Yeast Plasmid Miniprep II (from Zymo Research)
  • Zymolyase
  • solution 1
  • solution 2
  • solution 3
  • wash buffer

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