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.

Aliquot 5 mL of culture to a 15 mL falcon tube and centrifuge 3000x g

Pour off supernatant and pipette out the rest.

Resuspend the cells in 1 mL 0.9% w/v NaCl solution

Centrifuge e 3000x g , discard supernatant

Resuspend in 500µL sorbitol buffer

Sorbitol buffer:

1M Sorbitol

0.1M Na2EDTA

Add 20µL of zymolyase solution

Zymolyase solution:

Zymolyase 20T (12.5 mg/mL) or Zymolyase 100T (2.5 mg/mL) in Sorbitol buffer

Incubate 37°C 1h 0m 0s with gentle shaking

Centrifuge e 3000x g , discard supernatant

Resuspend the cells in 500µL yeast resuspension buffer

Yeast resuspension buffer:

50 mM Tris-HCl pH 7.5

20 mM Na2EDTA

Add 50µL 10% w/vSDS and shake vigorously

Add 5µL of 20mg/mL Proteinase K

Note: NEB proteinase K solution comes at this concentration

Incubate at 65°C for 0h 30m 0s

Add 200µL of 5M potassium acetate

Incubate On ice 0h 30m 0s

Pellet by centrifugation at max speed for 0h 5m 0s

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.

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.

Recover supernatant in a fresh microcentrifuge tube. Aim to get 500<x<700 uL of sample, and avoid getting debris.

Add pure isopropanol equal to the volume of the sample (~765µL ) and mix gently

Incubate for 0h 5m 0s``Room temperature

Note: do not allow more than 5 minutes to pass

Centrifuge at max speed for 0h 10m 0s

Discard supernatant

Air dry the pellet 0h 10m 0s

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

Note: NEB RNAse A comes in this concentration

TE solution:

10mM Tris pH 7.4

1mM Na₂EDTA

Incubate 37°C 0h 30m 0s

Add 30µL 3M sodium acetate and mix by inversion

Add 200µL isopropanol and mix, then centrifuge at max speed for 0h 0m 20s

Discard supernatant

Allow pellet to air dry 0h 10m 0s

Resuspend in 50-150µL 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.

Quantify using NanoDrop. Remember to blank using TE instead of water.

DNA is clean enough for PCR or sequencing.

Optional step for p1 analysis

Cast a 0.75% agarose 1X TAE gel

Load ~ 1µg 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)