EGFP was previously cloned into a lentiviral vector with the constitutive human EF-1α promoter (Addgene plasmid 12250)59 (link),61 (link). Lentivirus was produced in HEK293T/17 (ATCC CRL-11268, Manassas, VA), concentrated ∼75-fold using Amicon Ultra 100 kDa MWCO filters (Millipore, Cork, Ireland), and titered as described previously59 (link).
Amicon ultra 100 kda mwco filters
Manufactured by Merck Group
Sourced in Ireland, Germany
The Amicon Ultra 100 kDa MWCO filters are centrifugal filter devices used for the concentration and purification of macromolecules, such as proteins, peptides, and nucleic acids. The filters have a molecular weight cutoff (MWCO) of 100 kDa, which allows the selective retention of molecules larger than 100 kDa while allowing smaller molecules to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t 17, by American Type Culture Collection (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Econo pac 10dg desalting column, by Bio-Rad (1 mentions)
Alexa fluor 488 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
Fp 8500 spectrofluorometer, by Jasco (1 mentions)
6 protocols using amicon ultra 100 kda mwco filters
1
Lentiviral Vector Production for EGFP
2
Concentration and Resuspension of LPHNP
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For the characterization of the different formulations, the LPHNP suspensions were concentrated by ultrafiltration using Amicon® Ultra 100 KDa MWCO filters (Millipore, USA) at 10,000 rpm for 10 min and were resuspended to a final volume of 0.4 ml.
3
Lentiviral Vector Immobilization and Transduction
4
Doxycycline-inducible BMP2 Lentiviral Vector
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The doxycycline-inducible lentiviral vector was generated by cloning BMP2 into the modified TMPrtTA vector (provided by Danos Lab) as previously described (Rowland et al., 2018 (link)). To produce the lentivirus, HEK293T cells were co-transfected with either the dox-BMP2 vector or a constitutive GFP lentiviral vector with a second-generation packaging plasmid psPAX2 (Addgene, No. 12260) and an envelope plasmid pMD2.G (Addgene, No. 12259) by calcium phosphate precipitation (Salmon and Trono, 2007 (link)). The lentivirus was stored at –80°C until further use. The functional titer of each virus was determined via qRT-PCR to ascertain the number of lentiviral DNA copies integrated into the genome of the transduced HeLa cells (Salmon and Trono, 2007 (link)). On the day of transduction, the lentivirus was thawed and concentrated ∼100-fold using Amicon Ultra 100 kDa MWCO filters (Millipore, UFC9100).
5
Fluorescent Labeling of Bacterial Proteins
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For labeling of PomX-His6-Cys the protein-containing fractions were mixed with 125 µg Alexa Fluor 488 or 647 C5 Maleimide (Thermo Fisher Scientific) and incubated for 2 h at RT under denaturing conditions. Subsequently, the unreacted dye was removed by repeated concentration and dilution in Amicon Ultra 100 kDa MWCO filters (Merck Millipore, Darmstadt, Germany) using dialysis buffer (50 mM HEPES/NaOH, 50 mM KCl, 0.1 mM EDTA, 6 M urea, 2.5% (v/v) glycerol, pH 7.2) at 4 °C. Afterward, the protein was refolded via dialysis as described above. The labeled protein is indicated as PomX-A488 or PomX-A647. Cys-FtsZ contains an N-terminal MGKCKGSG extension to FtsZ. For labeling of Cys-FtsZ, the protein was mixed with 125 µg Alexa Fluor 488 C5 Maleimide (Thermo Fisher Scientific) for 2 h at 4 °C before unreacted dye was separated from the protein using an Econo-Pac 10DG desalting column (Biorad, Hercules, USA).
6
DNA Origami Folding and Purification
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DNA origami produced throughout this work was based on a previous design (named origami L, Linear) described elsewhere. 68 Briefly, folding of the DNA origami structures was performed in a one-pot reaction mix. 200 nM staple oligonucleotides were mixed with 20 nM p7249 plasmid in a folding buffer containing 5 mM Tris-HCl, 1 mM EDTA, 20 mM MgCl 2 and pH 8.0 (1 Â FOB20). Thermal annealing was subsequently performed from 65 to 60 1C in 1 h and from 59 to 40 1C in 40 h, on a Eppendorf Mastercycle Pro (Hamburg, Germany) thermal cycler. Purification of the folded structures (in order to remove the excess of staple strands) was done using size-exclusion centrifugal filtration with Amicon Ultra 100 kDa MWCO filters (Merck Millipore, Darmstadt, Germany) with an experimental buffer consisting of 5 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl 2 , 300 mM NaCl, pH 8.0. Bulk concentrations of the purified fluorescently-labelled DNA origami structures were finally determined using a Jasco FP-8500 spectrofluorometer (Tokyo, Japan).
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