Genetic complexity of living systems

Get prepared

You need to purchase Genomic-tip 500/G columns from QIAGEN (ref 10262), with the Genomic DNA Buffer Set from QIAGEN (ref 19060), Zymolyase (ref 120491 from SEIKAGAKU distributed by MPbiomedicals) and Proteinase K.

Sorbitol Buffer Y1: (should be included in the QIAGEN Buffer set, without the β-mercaptoethanol)
Sorbitol (1M final)                        91 g
EDTA 0.5M (100 mM final)            100 ml
β-mercaptoethanol (14mM final)     500 μl          ## FUME HOOD! ##
H2O:                                    qsp 500 ml
Store at 4°C.
Zymolyase stock:
Store aliquots of 1000 U in powder at 4°C (NOTE: Activity drops if stored in solution, even at -80°C)

Proteinase K stock:   20mg/ml in H2O (store in 400μl at -20°C)
RNaseA Stock : 30mg/ml in H2O, BOIL for 10min, store in 100μl aliquots at -20°C.


Let's do it

Grow 200ml YPD cultures from a colony overnight.

Estimate cell density (1 OD600  ~  107 cells/ml). Pellet between 0.5 to 2.10¹⁰ (Not more!) cells by centrifugating at 4000 rpm for 5min in Beckmann Allegra 25R ( = 3400 g).

Resuspend in 12ml TE and re-centrifuge similarly.

Resuspend cells in 12ml Sorbitol Buffer Y1. Resuspend 1000U of zymolyase in 1ml Sorbitol Buffer Y1 and add to the cells. Incubate at 30°C for 3hours (with shaking or swirling).

Check cell-wall lysis: Put 2 cell droplets on a microscope slide, add 0.1% SDS into one of them and compare the two situations under the microscope: speroplasts must be lysed by SDS.

Pellet spheroplasts by centrifugating at 5000 g, 4°C, for 10min.

Add 30 μl of RnaseA stock to 15ml of buffer G2. Mix. Pour on the cell pellet and resuspend thoroughly by vortexing.

Note: Prepare now centrifuge GS-15R by setting its rotor C0650 and refrigerating at 4°C. This way, the rotor will be cold when you'll need it.

Add 400μl of ProteinaseK stock solution. Incubate for at least 30minutes at 50°C.

Centrifuge at 5000g, 4°C, 10min. Meanwhile equilibrate a column with 10ml QBT buffer. Transfer the supernatant to a fresh 50ml Falcon tube, centrifuge again at 5000g, 4°C, 10min.

Transfer supernatant to a fresh 50ml Falcon tube, Vortex like hell and load on column.

Wash twice with 15ml QC. Meanwhile, pre-warm QF buffer at 50°C.

Elute with 15ml warm QF.

Add 10.5 ml isopropanol (Room Temp), mix by invertion. Centrifuge at 8000g for 15min at 4°C.

Rinse the pellet with 4ml cold (-20°C) ethanol 70% solution. Centrifuge at 8000g for 10min at 4°C on Beckman GS-15R (cold rotor)

Let dry briefly in the open tube at room temperature (not more than 15 minutes). Resuspend in 1ml TE.

Note: for Jouan GR412: 1850g = 3000rpm, 3000g = 4000 rpm, 4500g = 5000 rpm.