Preparation of bacterial cell lysate for SDS-PAGE (2022 iGEM)

Grow the cells overnight, to an OD₆₀₀ above 1. If IPTG induction needed, at it around OD₆₀₀ 0.2-0.3 (keep IPTG stock solution at -20 freezer).

Take one 1 ml of OD₆₀₀ = 1 from each sample. Correct the sample volume to obtain an equivalent size pellet. For us, as quantified using NanoCym950 nanoparticles (equivalent to the size of E. coli ), 1 OD600 equals to 10^8 nanoparticles per mL. We estimate each well on SDS-PAGE contains proteins from 2*10^6 bacteria.

Spin down the bacterial cells for 1 min at 13,000 rpm in a microfuge.

Aspirate any trace of supernatant with a vacuum line avoiding touching the pellet, but removing all liquid.

To each cell pellet, add 250 µL of distilled water, resuspend the pellet. Add 250 µL 2x SDS sample buffer (see below). Heat with a 95 degree heating block for 10 minutes. Store the samples at -20 degree freezer until SDS-PAGE.

2x SDS sample buffer, works for Tris-Cl or MOPS based gels

• Glycerol 20 ml
• SDS 4 g
• 0.5 M Tris-HCl pH 6.8 stock solution 25 ml
• Bromo Phenol Blue 1 mg; dissolve and volume to 95 ml
• add 5% β-Mercaptoethanol before usage