Bio-Rad MicroRotofor™ Cell Lysis Kits User Manual
Page 9
13. Add 600 µl of freshly prepared PSB solution to the
spheroplast pellet.
14. Sonicate the suspension to break down the cell
membrane and the genomic DNA. Sonication should
be performed in an ice bath to prevent heating.
Sonication should be performed with bursts of
20–30 sec, with chilling of the suspension on ice
between bursts.
15. Centrifuge at 20,000 x g for 30 min at 20°C and collect
the clear lysate.
16. Resuspend the residual cell debris in 250 µl of PSB
solution. Sonicate the suspension once briefly. Repeat
the centrifugation in step 15, collect the supernatant,
and pool with the first supernatant.
17. Determine the protein concentration of the extract.
This is best done using the RC DC protein assay
(catalog #500-0121 or 500-0122), which is compatible
with the detergents and reducing agents in PSB. If
performing the RC DC protein assay, keep in mind that
two washes of the sample are recommended. (Optional:
A reduction and alkylation of the sample is recommended
at this point in the procedure. Refer to the ReadyPrep
reduction-alkylation kit, catalog #163-2090.) Store the
protein extract at –70°C, apply directly onto an IPG
strip (see Appendix for recommendations), or proceed
to step 18.
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