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Cryo-EM sample preparation

BL Byung-Gil Lee
JL Jan Löwe

Last updated date: Sep 30, 2020 Views: 1136 Forks: 0

original An abbreviated version of this protocol was published in eLife in Sep, 2020
Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3
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1 μM purified S. cerevisiae cohesin EQ/EQ trimer (Smc1E1158Q, Smc3E1155Q, Scc1-2xStrepII) was incubated for 30 min at 4˚C with 1 μM Scc2C2(151-1493), forming cohesin tetramer, 5 mM ATP, and 1.3 μM of a 40 bp dsDNA (5’-GAATTCGGTGCGCATAATGTATATTATGTTAAATAAGCTT-3’, 5’-AAGCTTATTTAACATAA1055TATACATTATGCGCACCGAATTC-3’) or relaxed      plasmid DNA, in buffer (30 mM Tris-HCl, 60 mM NaCl, 1 mM TCEP, 5 mM MgCl2 pH 7.5). No cross-linking reagent was used. The plasmid was 1789 bp, derived from pUC19, containing a single site for Nt.BspQI nicking endonuclease. Nicking was performed according to the manufacturer’s instructions for 60 min at 50 °C (NEB) and the product was purified using a PCR purification kit (Qiagen) and eluted in water. For vitrification, 3 μl of sample were applied to glow-discharged Quantifoil Au 2/2 holey carbon 200 mesh grids or Ultrafoil Au 2/2 holey gold 200 mesh grids (Quantifoil). The grids were blotted for 3s at 4˚C and 100%  humidity with a blotting force of level -15, and flash frozen in liquid ethane using an FEI Vitrobot Mark IV (Thermo Fisher Scientific) and a liquid-ethane cryostat set to −180 °C (Russo et al., 2016).
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Lee, B and Löwe, J(2020). Cryo-EM sample preparation. Bio-protocol Preprint. bio-protocol.org/prep527.
  2. Collier, J. E., Lee, B., Roig, M. B., Yatskevich, S., Petela, N. J., Metson, J., Voulgaris, M., Gonzalez Llamazares, A., Löwe, J. and Nasmyth, K. A.(2020). Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3. eLife. DOI: 10.7554/eLife.59560

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