Journal: Current protocols in molecular biology

Article Title: Virion Display: A High-Throughput Method to Express Functional Membrane Proteins

doi: 10.1002/cpmb.126

Figure Lengend Snippet: Overview of the cloning steps for VirD (Basic Protocol 1). a. The first step of the Gateway cloning protocol is to attach attB sequences (and remove STOP codon, if applicable) to a given ORF via PCR. This PCR product is then shuttled into a Gateway entry vector via the BP reaction. b. The next step of the Gateway cloning process is to insert the ORF ΔSTOP currently in the entry vector into the HSV-1 ΔUL27 CV5 Gateway BAC destination vector via the LR reaction.

Article Snippet: Materials: Q52X High-Fidelity Master mix (New England BioLabs, Catalog #: M0492S) DMSO Gel 6x loading dye (NEB Catalog #: B7024S) 1kp plus DNA ladder (Thermo Fisher Scientific, Catalog #: 10787018) 1x TTE Buffer (500mM Tris-base, 500-mM TAPS acid, and 10mM EDTA) Agarose Ethidium Bromide Qiaquick gel purification kit (Qiagen, Cat No./ID: 28704) 3M Sodium Acetate, pH 5.2 Isopropanol TE buffer (Thermo Fisher Catalog #: 12090015) list-behavior=simple prefix-word= mark-type=none max-label-size=0 Alternatively: recipe is 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA pDONR221 (Thermo Fisher Scientific Catalog #: 12536017) BP Clonase II Enzyme (Thermo Fisher Scientific Catalog #: 11789020) 0.2 cm cuvettes (Bio-Rad, Catalog #: 1652082) DH5α cells (Thermo Fischer Scientific, Catalog #: 18265017) TOP10 cells (Thermo Fischer Scientific, Catalog #: C404050) One Shot™ ccdB Survival™ 2 T1R Competent Cells (Thermo Fisher Scientific, Catalog #: A10460) SOC Media 2YT Medium 2YT 2% Agar plates Kanamycin QIAprep Spin Miniprep Kit (250) (Qiagen, Cat No./ID: 27106) Bsr GI Restriction Enzyme (New England BioLabs Catalog #: R0575S) list-behavior=simple prefix-word= mark-type=none max-label-size=0 Enzyme is either supplied in the appropriate buffer or includes 10X NEBuffer 2.1 HSV-1 ΔUL27 CV5 Gateway BAC (available from Heng Zhu’s or Prashant Desai’s lab) LR Clonase II Enzyme (Thermo Fisher Scientific Catalog #: 11791020) Phire 2X Master Mix Hot Start Polymerase (Thermo Fisher Scientific Catalog #: F125S) Deep 96 well boxes (both round bottom and v bottom) 96-well Aluminum foil seals 0.2 mL PCR strip tubes 1.5 mL Microcentrifuge Tubes Round Bottom Culture Tubes Bio-Rad Hot lid thermal cycler Incubator Rotator Bio-Rad Gene Pulser Xcell Electroporation System Tabletop microcentrifuge Protocol Steps: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 1.

Techniques: Clone Assay, Plasmid Preparation

Journal: Current protocols in molecular biology

Article Title: Virion Display: A High-Throughput Method to Express Functional Membrane Proteins

doi: 10.1002/cpmb.126

Figure Lengend Snippet:

Article Snippet: Materials: Q52X High-Fidelity Master mix (New England BioLabs, Catalog #: M0492S) DMSO Gel 6x loading dye (NEB Catalog #: B7024S) 1kp plus DNA ladder (Thermo Fisher Scientific, Catalog #: 10787018) 1x TTE Buffer (500mM Tris-base, 500-mM TAPS acid, and 10mM EDTA) Agarose Ethidium Bromide Qiaquick gel purification kit (Qiagen, Cat No./ID: 28704) 3M Sodium Acetate, pH 5.2 Isopropanol TE buffer (Thermo Fisher Catalog #: 12090015) list-behavior=simple prefix-word= mark-type=none max-label-size=0 Alternatively: recipe is 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA pDONR221 (Thermo Fisher Scientific Catalog #: 12536017) BP Clonase II Enzyme (Thermo Fisher Scientific Catalog #: 11789020) 0.2 cm cuvettes (Bio-Rad, Catalog #: 1652082) DH5α cells (Thermo Fischer Scientific, Catalog #: 18265017) TOP10 cells (Thermo Fischer Scientific, Catalog #: C404050) One Shot™ ccdB Survival™ 2 T1R Competent Cells (Thermo Fisher Scientific, Catalog #: A10460) SOC Media 2YT Medium 2YT 2% Agar plates Kanamycin QIAprep Spin Miniprep Kit (250) (Qiagen, Cat No./ID: 27106) Bsr GI Restriction Enzyme (New England BioLabs Catalog #: R0575S) list-behavior=simple prefix-word= mark-type=none max-label-size=0 Enzyme is either supplied in the appropriate buffer or includes 10X NEBuffer 2.1 HSV-1 ΔUL27 CV5 Gateway BAC (available from Heng Zhu’s or Prashant Desai’s lab) LR Clonase II Enzyme (Thermo Fisher Scientific Catalog #: 11791020) Phire 2X Master Mix Hot Start Polymerase (Thermo Fisher Scientific Catalog #: F125S) Deep 96 well boxes (both round bottom and v bottom) 96-well Aluminum foil seals 0.2 mL PCR strip tubes 1.5 mL Microcentrifuge Tubes Round Bottom Culture Tubes Bio-Rad Hot lid thermal cycler Incubator Rotator Bio-Rad Gene Pulser Xcell Electroporation System Tabletop microcentrifuge Protocol Steps: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 1.

Techniques: