TITLE: Yeast gDNA isolation
AUTHORS: is Sparrow

[DESCRIPTION]
This protocol is based on https://cshprotocols.cshlp.org/content/2020/10/pdb.prot098152.full doi:10.1101/pdb.prot098152 

The protocol details how to perform a small scale yeast gDNA isolation including a proteinase K incubation step to visualize linear cytoplasmic protein primed plasmids. 

This is used in combination with the system OrthoRep which relies on error prone replication of linear cytoplasmic plasmids.
Rix, G., Watkins-Dulaney, E.J., Almhjell, P.J. et al. Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities. Nat Commun 11, 5644 (2020). https://doi.org/10.1038/s41467-020-19539-6

[STEPS]
SECTION: DNA extraction
1. Start a 10 mL yeast culture and grow to saturation overnight or over 2 days

Note: for p1_wt (but not p1_rec) containing strains, p1 can be lost if grown in YPD for prolonged periods of time (based on my estimations, 2 or 3 1:1000 passages). Be mindful of this.
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2. Aliquot 5 mL of culture to a 15 mL falcon tube and centrifuge 3000 x g, 5 min 

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3. Pour off supernatant and pipette out the rest.
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4. Resuspend the cells in 1 mL 0.9 % w/v NaCl solution

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5. Centrifuge e 3000 x g, 2 min , discard supernatant
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6. Resuspend in 500 µLsorbitol buffer 

Sorbitol buffer:

1M Sorbitol
0.1M Na2EDTA

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7. Add 20 µL of zymolyase solution

Zymolyase solution:
Zymolyase 20T (12.5 mg/mL) or Zymolyase 100T (2.5 mg/mL) in Sorbitol buffer
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8. Incubate 37 °C 60 min with gentle shaking

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9. Centrifuge e 3000 x g, 5 min , discard supernatant
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10. Resuspend the cells in 500 µL yeast resuspension buffer

Yeast resuspension buffer:
50 mM Tris-HCl pH 7.5
20 mM Na2EDTA 

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11. Add 50 µL 10 % w/vSDS and shake vigorously

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12. Add 5 µL of 20 mg/mL Proteinase K

Note: NEB proteinase K solution comes at this concentration

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13. Incubate at 65 °C for 30 min

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14. Add 200 µL of 5M potassium acetate

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15. Incubate on ice 30 min 

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16. Pellet by centrifugation at max speed for 5 min


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17. Recover supernatant in a fresh microcentrifuge tube. Aim to get 500<x<700 uL of sample, and avoid getting debris. 
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18. Add pure isopropanol equal to the volume of the sample (~765 µL ) and mix gently

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19. Incubate for 5 minRoom temperature 

Note: do not allow more than 5 minutes to pass

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20. Centrifuge at max speed for 10 min 

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21. Discard supernatant
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22. Air dry the pellet 10 min 

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23. Add 300 µL of TE solution and add 0.75 µL of RNAse A at 20 mg/mL 

Note: NEB RNAse A comes in this concentration

TE solution: 
10mM Tris pH 7.4
1mM Na2EDTA
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24. Incubate 37 °C 30 min 
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25. Add 30 µL 3M sodium acetate and mix by inversion

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26. Add 200 µL isopropanol and mix, then centrifuge at max speed for 20 s  

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27. Discard supernatant
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28. Allow pellet to air dry 10 min 

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29. Resuspend in 50-150 µL TE solution

TE solution: 
10mM Tris pH 7.4
1mM Na2EDTA

Pellet might be hard to resuspend especially if there is a lot of DNA. Be gentle, but persistent. 
SECTION: DNA isolation
30. Quantify using NanoDrop. Remember to blank using TE instead of water.

DNA is clean enough for PCR or sequencing. 
SECTION: Gel analysis
31. Optional step for p1 analysis

Cast a 0.75% agarose 1X TAE gel

Load ~ 1 µg gDNA 

Run at 85 V for 100+ minutes

Look for the following bands and no others

p2_wt: 13.5 kb

p1_wt (size will be variable depending on landing pad used - for GA-Y319 = 8.9 kb; for GR-Y718 = 5.6 kb)

p1_rec (size will be variable = your construct size + 4.6 kb)
SECTION: DNA isolation
16.1. Note:

At this step, a "short" protocol can be done - it will not produce sufficient DNA for visualization of p1, but is sufficient for PCR amplification from p1.

SECTION: DNA isolation
16.2. Recover supernatant and place into a miniprep kit silica column. Wash as per manufacturer instructions or with homemade Wash 1 / Wash 2 buffers, and elute in water or EB. 

Note: this short protocol is not suitable for electrophoresis visualization of gDNA and should only be used for PCR amplification from the eluted DNA.