Journal: STAR Protocols

Article Title: Protocol for the recovery and deep sequencing of short ssDNA pools from transient, fuel-dependent coacervate droplets

doi: 10.1016/j.xpro.2025.104293

Figure Lengend Snippet: ssDNA isolation and library preparation (A) Prior to the addition of fuel, the peptide, polyU RNA and ssDNA molecules are mixed in solution, and do not form supramolecular assemblies. When fuel (EDC) is added, phase separation occurs, forming coacervate droplets that sequester the ssDNA molecules from the polymer-poor phase. The phases are separated via centrifugation to yield the polymer-poor and polymer-rich phases, from which the ssDNA populations are isolated. For a complete characterization of this droplet system, please refer to Donau et al., 2020. (B) Library preparation relies on the orientation-specific ligation of two adapter molecules to the recovered ssDNA oligonucleotides. After the ligation, the sequencing primers are added in a PCR using primers with overhangs. (C) Urea-PAGE is performed following library preparation. R = Reference, S = Supernatant, D = Droplets. ‘Input’ corresponds to the samples obtained from the droplets, while the ‘R’ sample is the naïve input library. ‘Ligation’ corresponds to the product of the first step in B and shows a clear band (red star) corresponding to the expected full-length ligation product, as well as a weaker smear (green stars) corresponding to the two partially adapter-ligated products. Bands at 45 nt correspond to the adapters and splint, and bands at ∼30 correspond to unreacted input ssDNA. ‘Product’ shows the full-length sequencing library, with the correct PCR band being marked with a red star. Several additional bands are visible, including two prominent bands (blue stars), which are located approximately 30 nt above and below the main PCR band. These correspond to the no-insert (adapter dimer) and double-insert ligation products. The smear and bands below correspond to indexing primers. (E) Electropherogram of a TapeStation run of a prepared library. Only the peak corresponding to the correct library size (∼160 bp) is used to calculate the correct library dilution for loading onto the sequencer.

Article Snippet: Monarch Spin PCR & DNA Cleanup Kit , New England Biolabs , T1130L.

Techniques: Isolation, Polymer, Centrifugation, Ligation, Sequencing

Journal: Nucleic Acids Research

Article Title: Integrase-On-Demand : bioprospecting integrases for targeted genomic insertion of genetic cargo

doi: 10.1093/nar/gkag106

Figure Lengend Snippet: Functional integrases mediate site-specific recombination between predicted attB sites. (A) To verify recombination occurs at the predicted chromosomal attB and plasmid attP sites, DNA from attR -containing junctions were PCR amplified using a primer annealing to chromosomal DNA and a primer annealing to plasmid DNA. (B–G) PCR products using wild-type P. putida S12 (WT) or Tc R P. putida S12 colonies post electroporation of int genes encoding (B) Pal_S-Int_1, (C) Pal_Y-Int_1, (D) Pal_Y-Int_3, (E) Ppu_Y-Int_1, (F) Pal_S-Int_1, (G) Ppu_Y-Int_4, and cognate attP containing test plasmids.

Article Snippet: To generate the positive control plasmid pUCP22- tcR, tcR (conferring tetracycline resistance) from pACYC184 was PCR amplified with Q5 DNA polymerase (NEB, Ipswich, MA, M0491S) and cloned into pUCP22 using restriction cloning (Eco53kI (NEB, Ipswich, MA, R0116S), XbaI (NEB, Ipswich, MA, R0145S), Monarch ® Spin PCR & DNA Cleanup Kit (NEB, Ipswich, MA, T1130L), Ligase (NEB, Ipswich, MA, M0202L). pUCP22- tcR was maintained in NEB 5-alpha E. coli with tetracycline selection.

Techniques: Functional Assay, Plasmid Preparation, Amplification, Electroporation

Journal: Nucleic Acids Research

Article Title: Integrase-On-Demand : bioprospecting integrases for targeted genomic insertion of genetic cargo

doi: 10.1093/nar/gkag106

Figure Lengend Snippet: Testing Sel_Y-Int_1 integration at native cyanobacterial S. elongatus attB site. (A) Schematic of the kanamycin resistance-conferring, nonreplicating plasmid bearing an R6K ori, origin of transfer for conjugation (oriT), the Sel_Y-Int_1-encoding int driven by the lambda phage pR promoter, and the cognate attP site. (B) Schematic of integrase-mediated recombination between the plasmid attP site and the many copies of the chromosomal attB site, which leads to stable kanamycin resistance. (C) Km R clones obtained were assayed for integration via PCR of the attB site. “Integrated” clones would have yielded only a band detecting the integrated plasmid, whereas “unsegregated” clones displayed both wild-type “WT” and integrated loci. WT clones would have displayed amplification for only the undisrupted locus, but no such instances were detected.

Article Snippet: To generate the positive control plasmid pUCP22- tcR, tcR (conferring tetracycline resistance) from pACYC184 was PCR amplified with Q5 DNA polymerase (NEB, Ipswich, MA, M0491S) and cloned into pUCP22 using restriction cloning (Eco53kI (NEB, Ipswich, MA, R0116S), XbaI (NEB, Ipswich, MA, R0145S), Monarch ® Spin PCR & DNA Cleanup Kit (NEB, Ipswich, MA, T1130L), Ligase (NEB, Ipswich, MA, M0202L). pUCP22- tcR was maintained in NEB 5-alpha E. coli with tetracycline selection.

Techniques: Plasmid Preparation, Conjugation Assay, Clone Assay, Amplification