Yeast gDNA isolation
is Sparrow
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Abstract
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
DNA extraction
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
DNA isolation
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.
Gel analysis
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)
