Rhodobacter 1558 lyse ficoll

Description

Technical info

Tilt Series Date: 2013-09-26

Data Taken By: Ariane Briegel

Species / Specimen: Rhodobacter sphaeroides

Strain: ATCC 17023

Tilt Series Settings: Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 160.0 eV/Ų, defocus: -12.0 μm, magnification: 22500x.

Microscope: Caltech Polara

Acquisition Software: UCSFTomo

Upload Method: pipeline

Processing Software Used: Raptor

Collaborators and Roles: Judy Armitage

Purification / Growth Conditions / Treatment: Spun down for 10' at 5100 rpm. Resuspended pellets in 5 mL PEG-Trehalose-Ficoll-Sucrose-Tris-HCl buffer (7.5% PEG 8000, 4% Trehalose, 20% Sucrose, 25 mM Tris-HCl pH 7.0,10% Ficoll) * 2.14 mL PEG 8000 * 1 mL Trehalose * 2 g Sucrose * 1 g Ficoll * Q.S. 10 mL 25 mM Tris-HCl, pH 7.0Note: Ficoll didn't go into solution after 4 hours, so spun down and used aqueous phase. In future, try less Ficoll?Add 500 µL of 50 mg/mL lysozyme stock (make fresh, in 10 mM Tris-HCl, pH 8.0) for a final concentration of 5 mg/mL Incubate 15' at room temperature Add 28 µL of magnesium chloride, 3.3 µL of calcium chloride, and 5.5 µL of DNase IIncubate 30' at 37 degrees Celsius Spin down for 10' at 5100 rpm.Resuspend with gentle pipetting in 200 µL K-phosphate-PEG-Trehalose-Sucrose buffer (7.5% PEG-8000, 4% Trehalose, 20% Sucrose, 5 mM K-phosphate buffer) * 2.14 mL PEG 8000 * 1 mL Trehalose * 2 g Sucrose * Q.S. 10 mL 5 mM K-phosphate buffer, pH 7.0 9/17/13: Seems to work well. Good spheroplast formation, and intact clusters.Freeze grids in grid box "Waldo Ficoll"

Attached Files

Waldo22mag160dose0004.mrc, Tilt Series (Pixel Size 0.48 nm), 3.4 GB