Pet28a cas9 cys

Manufactured by Addgene

Sourced in United States

The PET28a/Cas9-Cys is a plasmid vector that expresses the Cas9 protein with a cysteine tag. It is commonly used in CRISPR-Cas9 gene editing experiments.

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Lab products found in correlation

Hiprep 26 10 desalting column, by GE Healthcare (2 mentions) Histrap hp 5 ml column, by GE Healthcare (2 mentions) Bl21 de3 cells, by New England Biolabs (2 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)

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PET28a (13 citations)

2 protocols using pet28a cas9 cys

Purification of CRISPR-Cas9 Protein

Cited 1 time

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pET28a/Cas9-Cys was a gift from Hyongbum Kim (Ramakrishna et al., 2014 (link)). BL21(DE3) cells (New England Biolabs, C2527) were transformed with the plasmid pET28a/Cas9-Cys (Addgene, Cambridge, USA, plasmid #53261) using standard protocols. Cas9 protein expression was induced with 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) (Fisher, 10715114) and bacterial cells were incubated overnight at 20°C. Bacterial pellets were resuspended in 20 mL of lysis buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM TCEP, 5 mM imidazole pH 8.0), sonicated and loaded on a HisTrap HP 5 mL column (GE, 17-5248-01). Cas9 protein was collected in elution buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 10% glycerol, 1 mM TCEP, 250 mM imidazole pH 8.0). Fractions containing Cas9 protein were pooled and loaded into a HiPrep 26/10 Desalting Column (GE, 28-4026-52) to equilibrate in Cas9 buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM TCEP). Purified Cas9 protein was further concentrated using Vivaspin columns (Vivaspin20, VS2021) as per the user-guide instructions.

Gangoso E., Southgate B., Bradley L., Rus S., Galvez-Cancino F., McGivern N., Güç E., Kapourani C.A., Byron A., Ferguson K.M., Alfazema N., Morrison G., Grant V., Blin C., Sou I., Marques-Torrejon M.A., Conde L., Parrinello S., Herrero J., Beck S., Brandner S., Brennan P.M., Bertone P., Pollard J.W., Quezada S.A., Sproul D., Frame M.C., Serrels A, & Pollard S.M. (2021). Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion. Cell, 184(9), 2454-2470.e26.

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Cas9 Protein Purification from BL21(DE3)

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BL21(DE3) cells (New England Biolabs, C2527) were transformed with the plasmid pET28a/Cas9-Cys (Addgene, Cambridge, USA, plasmid #53261) using standard protocols. Cas9 protein expression was induced with 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) (Fisher, 10715114) and the cells were incubated overnight at 20°C. 24 hr later, bacterial pellets were resuspended in 20 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM TCEP, 5 mM imidazole pH 8.0), sonicated and loaded on a HisTrap HP 5 ml column (GE, 17-5248-01). The Cas9 protein was collected in elution buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 10% glycerol, 1 mM TCEP, 250 mM imidazole pH 8.0). The fractions containing Cas9 protein were pooled and loaded into a HiPrep 26/10 Desalting Column (GE, 28-4026-52) to equilibrate in Cas9 buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM TCEP). The purified Cas9 protein was further concentrated using Vivaspin columns (Vivaspin20, 30 000 MWCO PES, Sartorius stedim, VS2021) as per the users-guide instructions.

Dewari P.S., Southgate B., Mccarten K., Monogarov G., O'Duibhir E., Quinn N., Tyrer A., Leitner M.C., Plumb C., Kalantzaki M., Blin C., Finch R., Bressan R.B., Morrison G., Jacobi A.M., Behlke M.A., von Kriegsheim A., Tomlinson S., Krijgsveld J, & Pollard S.M. (2018). An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein. eLife, 7, e35069.

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