Journal: Current Protocols

Article Title: A Rapid Method to Rank Proteins of Widely Varying Stability: Application to Paramyxovirus Foot Domains

doi: 10.1002/cpz1.70479

Figure Lengend Snippet: Ligation‐independent cloning scheme. ( A ) Schematic diagram of the gene coding for the fusion construct on the vector suBLA. ( B ) LIC site with BseR1 recognition site indicated in bold and cut site indicated with triangles. Proximal cytosine is indicated in red. ( C ) Cleavage of vector by BseR1 disrupts stop codon. ( D ) Treatment of cleaved vector with T4 polymerase in presence of dCTP degrades 3’ ends at the cut site back to proximal cytosines, generating large overhangs. ( E ) PCR product of insert with LIC handles. Proximal guanine is shown in red. ( F ) Treatment of PCR product with T4 polymerase in presence of dGTP degrades 3’ ends back to proximal guanine, generating overhangs complementary to those on vector. ( G ) Insert annealed into vector. Successful insertion converts the stop codon at the BseR1 cut site to TCT, coding for serine.

Article Snippet: suBLA vector miniprep (see Strategic Planning) 10× restriction enzyme buffer (e.g., rCutSmart Buffer, New England Biolabs, cat. no. B6004S, supplied with BSER1) H 2 O, RNase‐free BseR1 restriction enzyme (e.g., New England Biolabs, cat. no. R0581S) Alkaline phosphatase, calf or shrimp (e.g., New England Biolabs, cat. nos.

Techniques: Ligation, Cloning, Construct, Plasmid Preparation

Journal: Current Protocols

Article Title: A Rapid Method to Rank Proteins of Widely Varying Stability: Application to Paramyxovirus Foot Domains

doi: 10.1002/cpz1.70479

Figure Lengend Snippet: Troubleshooting Guide for Ligation‐Independent Cloning

Article Snippet: suBLA vector miniprep (see Strategic Planning) 10× restriction enzyme buffer (e.g., rCutSmart Buffer, New England Biolabs, cat. no. B6004S, supplied with BSER1) H 2 O, RNase‐free BseR1 restriction enzyme (e.g., New England Biolabs, cat. no. R0581S) Alkaline phosphatase, calf or shrimp (e.g., New England Biolabs, cat. nos.

Techniques: Ligation, Cloning, Plasmid Preparation, Control

Journal: Current Protocols

Article Title: Endogenous Protein Tagging in Postmitotic Neurons via Lentiviral and Adeno‐associated Virus Delivery

doi: 10.1002/cpz1.70492

Figure Lengend Snippet: Schematic of the two‐vector system: lentiviral pLentiSIV HA‐NLS‐SpCas9‐NLS and AAV1 encoding sgRNA, mEmerald donor, and mTagBFP2‐CAAX2 reporter. (A) pLentiSIV hSyn HA‐NLS‐SpCas9‐NLS WPRE: replication‐incompetent SIV‐based lentiviral backbone allowing the lentiviral expression of SpCas9 (Addgene plasmid #236242). (B) The pAAV KI Cloning Vector_hSyn mTagBFP2‐CAAX2: AAV backbone with U6 promoter, BbsI sgRNA cloning sites, gRNA scaffold, HindIII/XhoI donor insertion sites, and hSyn‐mTagBFP2‐CAAX2 reporter (Addgene plasmid #236245). (C) pAAV Jph3gRNA mEmerald hSyn mTagBFP2‐CAAX2 (Addgene ##236246) donor vector expressing sgRNA and donor sequence for the endogenous tagging of JPH3. Both flanking Cas9 target sites (T) must be the reverse complement of the genomic guide sequence (including PAM) to prevent Cas9 from re‐cutting a correctly integrated donor and to enforce directional integration via HITI. Parts of this figure were created with BioRender.com.

Article Snippet: pLentiSIV hSyn HA‐NLS‐SpCas9‐NLS WPRE (SIV‐based lentiviral backbone allowing the Lentiviral expression of SpCas9; Addgene plasmid # 236242 T4 DNA Ligase and Reaction Buffer (NEB, cat. no. M0202) T4 Polynucleotide Kinase (T4 PNK; NEB, cat. no. M0201) Oligonucleotides for sgRNA cloning (IDT; see protocol steps for sequences) BbsI‐HF restriction enzyme, (NEB, cat. no. R3539), HindIII‐ HF (NEB, cat. no. R3104), XhoI restriction enzymes (NEB, cat. no. R0146), and rCutSmart Buffer pAAV KI Cloning Vector: pAAV KI Cloning Vector_hSyn mTagBFP2‐CAAX2 (AAV backbone with U6 promoter, BbsI sgRNA cloning sites, gRNA scaffold, HindIII/XhoI donor insertion sites, and hSyn‐mTagBFP2‐CAAX2 reporter); Addgene plasmid #236245 Competent E. coli : DH5α (NEB, C2988J) or Stbl3 for viral plasmids (One Shot Stbl3 Chemically Competent E. coli , Invitrogen, cat. no. C737303) SOC medium (Sigma‐Aldrich, cat. no. S1797) LB agar plates with 100 μg/mL ampicillin (as described in Repository and Christensen, ) In‐Fusion HD Cloning Kit (Takara Bio, cat. no. 639648) or equivalent Gibson assembly kit Plasmid miniprep and maxiprep kits Annealing buffer: 10 mM Tris·HCl (pH 7.5), 50 mM NaCl, and 1 mM EDTA NEBNext Ultra II Q5 Master Mix (NEB, cat. no. M0544) Benchling molecular biology software ( https://benchling.com ) 37°C bacterial incubator with shaker Thermocycler UV spectrophotometer (NanoDrop or equivalent) Gel electrophoresis apparatus

Techniques: Plasmid Preparation, Expressing, Cloning, Sequencing