General considerations:
From step1, never use vortex. Until step 5, pipetting is not required. Then, use wide bore tips for pipetting. Always gently invert tubes for mixing. Minimize the movement when transferring from a tube to another one.
Prepare lysis buffer:
Always use fresh buffer: TrisHCl 114mM, EDTA 115mM, NaCl 571mM, 1.14% PVP40.
Add 0.5ml TrisHCl 1M, 0.5ml EDTA 0.5M, 0.5ml NaCl 5M, 0.05g PVP40 and 2.875ml Ultrapure nuclease-free water. Mix by vortexing.
Incubate 30min at 65°C. Filter sterilize the buffer.
Saccharomyces cerevisiae culture
Culture media is YPDA. Use only fresh (<4 weeks old) colonies. For pre-culture, one colony on an YPDA-Agar plate is dissolved in 5ml YPDA and grown for 5h at 30°C with shaking at 200rpm. Then 200ml YPDA is inoculated at a theoretical DO of 0,005 and grown for 16 hours (O/N) at 30°C 200rpm. Cultures are centrifuged in 50ml tubes for 5min at 1500g at 4°C. Pellet is dissolved in 20ml TE (10mM TrisHCl, 1mM EDTA) and centrifuged for 5min at 1500g at 4°C. Pellets are used fresh or can be flash frozen in liquid N2 and conserved at -80°C. One pellet contains approximately 1010 cells.
STEP1: Cell wall lysis
Resuspend a yeast pellet in 4ml Sorbitol 1M.
Add 250µl Zymolyase 1000U/ml. Invert gently 5x.
Incubate 30min at 30°C (Thermomixer C: program 10sec at 300rpm each 10min).
Centrifuge 2min30 at 3000g.
Discard supernatant, let the tube upside down to remove most of the liquid.
STEP2: Spheroplasts lysis
Resuspend the pellet in 3.5ml lysis buffer.
Add 500µl SDS 10%
Add 4µl RNAse A 100mg/ml
Invert gently 10-15x.
Incubate 30min at 50°C (Thermomixer C: program 10sec at 300rpm each 10min).
STEP3: Protein precipitation
Add 10ml TE
Add 5ml KAc 5M pH7.5
Invert gently 15x
Place on ice for 5min
Centrifuge 10min at 5000g at 4°C (acceleration 9, deceleration 9)
Transfer the supernatant in two 15ml tubes and repeat the last centrifugation.
STEP4: DNA precipitation
Transfer the two supernatants in one 50ml tube and add 1V Isopropanol at RT.
Invert gently about 15x. You shall see some DNA filaments at this stage.
Centrifuge 5min at 500g at 4°C (acceleration 9, deceleration 1)
Discard supernatant and dislodge pellet with 20ml ethanol 70%
Place on ice for at least 5min
Centrifuge 5min at 500g at 4°C (acceleration 9, deceleration 1)
Discard supernatant, remove as much ethanol as possible
STEP5: DNA elution
Let the pellet air dry for 5min
Add 200µl TE at 50°C
Incubate 10min at 50°C without cap in order to evaporate the remaining ethanol
Transfer carefully with wide bore tip in a 1.5ml tube.