Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome
for 5 minutes, and media was aspirated. Cells were then resuspended in the same volume of PBS , and the spin-down and aspiration was repeated to wash the cells and remove any IgG from serum. Dynabeads M-280 Protein G were resuspended by vortexing for 30 seconds. Then, 50 ml of blocking buffer was prepared by adding 1 g of biotin-free BSA and 200 μl of 0.5 M pH 8.0 EDTA into DPBS , vacuum filtering with a 0.22-μm filter and then kept on ice.
A library of mCherry amplicons with randomized barcodes after the stop codon was generated by PCR, electroporated into landing pad cells and recovered and sequenced from gDNA. More specifically, to construct the library, primers were designed to amplify the attP-mCherry-pA sequence off of the template plasmids used in previous landing pad assays , and the reverse primer included a randomized 6×N barcode as an extension. This primer was synthesized by IDT using standard mixed bases.
K562 landing pad clonal lines with the associated recombinase were then electroporated with these amplicon donors. In total, 1.2 × 10K562 cells were electroporated in 100 µl of Amaxa solution with either 250 ng or 750 ng of the amplicon donor. Seven days later, the efficiency of mCherry integration was determined by flow cytometry , and then, 1 day later, gDNA extraction was performed from 5 million cells using a Qiagen DNeasy Mini Prep Kit.
Australia Latest News, Australia Headlines
Similar News:You can also read news stories similar to this one that we have collected from other news sources.
A proteome-scale map of the SARS-CoV-2–human contactome - Nature BiotechnologyLandscape of molecular contacts: How SARS-CoV-2 communicates with human cells NatureBiotech
Read more »
Repairing the eye - Nature BiotechnologyRepairing the eye: Companies are pursuing interventions to edit, regenerate and reprogram eye tissue to restore lost vision
Read more »
Scientists Have Sequenced Dire Wolf DNA. Thanks, Science!Real dire wolves weren't just bigger wolves, they were 'the last surviving member of an ancient lineage distinct from all living canines.' No wonder they fit in so well on Game of Thrones.
Read more »
A comprehensive SARS-CoV-2–human protein–protein interactome reveals COVID-19 pathobiology and potential host therapeutic targets - Nature BiotechnologyA comprehensive SARS-CoV-2–human protein–protein interactome reveals COVID-19 pathobiology and potential host therapeutic targets
Read more »
Japan to Invest in Metaverse and NFT ExpansionJapan is diving deeper into the metaverse. Japan’s Prime Minister Fumio Kishida said the country will continue to focus on “supporting the social implementation of digital technology” and will “expand the use of Web3 services that utilize the metaverse.'
Read more »
Star Trek: Discovery Season 5 Trailer Released, Reveals New CastWatch the action-packed new trailer for StarTrekDiscovery Season 5, fresh from NYCC2022. StarTrek NYCC
Read more »