The CRISPR/Cas9 system was used to engineer ESCs for protein degradation of ZFP281, as previously described.52 (link) The 5’- and 3’-homology arms of Zfp281 for C-terminal insertion were PCR amplified from genomic DNA. The 5’- and 3’-homology arms and FKBP12F36V-2xHA-mCherry fragment were assembled by Gibson Assembly 2x Master Mix (NEB, E2611S). The CRISPR gRNA was subcloned into the pSpCas9(BB)-2A-Puro (PX459) vector (gRNA sequence in Table S3 ). ESCs were transfected with the donor and CRISPR vectors using Lipofectamine 3000 (Invitrogen). After 2 days of puromycin selection, mCherry-positive cells were seeded on a 96-well plate with single-cell per well using the BD Influx Cell Sorter. Cells were expanded and genotyped by PCR. Two clones (#2 and #21) with a homozygous knock-in were further expanded and used for experiments. Zfp281degron ESCs and cEpiSCs were treated with dTAG13 (500 nM in DMSO, Tocris, 6605) for degradation of ZFP281 protein.
Gibson assembly 2x master mix
Manufactured by New England Biolabs
Gibson Assembly 2x Master Mix is a pre-formulated solution for seamlessly joining multiple DNA fragments in a single reaction. The mix contains the necessary enzymes and reagents to facilitate the Gibson Assembly process, enabling efficient and reliable DNA assembly.
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Lab products found in correlation
4 protocols using gibson assembly 2x master mix
1
CRISPR-Mediated ZFP281 Degradation in ESCs
2
Circularization of RoC-ITS Product
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The linear RoC-ITS product was circularized with a DNA splint that matches the RoC-ITS 27F Unique and RoC-ITS 189r Unique primers using Gibson assembly [52 (link)]. In a 20 μl reaction, 150 ng of RoC-ITS product and splint DNA were combined with 10 μl of Gibson Assembly 2X Master mix (Catalog number: E2611S Vendor: New England Biolabs Inc) and incubated at 50 °C for 60 min. The reaction was bead-cleaned with 1.0x bead ratio (HighPrepTM PCR paramagnetic bead solution. Vendor: MAGBIO. Catalog number: AC-60050) and eluted in 42 μl of nuclease-free water. Linear molecules in the reaction mixture were then degraded using an exonuclease with the reaction carried out in a 50 μl volume: 2 µl of 25 mM ATP solution, 5 µl of 10X reaction buffer and 1 µl of Plasmid-Safe DNASe at 37 °C for 30 min. The circularized reaction product was isolated with a 1.0× bead cleanup and eluted in 12 μl of nuclease-free water.
3
Gibson Assembly for Plasmid Construction
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NEBuilder Assembly Tool was used to design primers for Gibson Assembly reactions. The fragments were amplified using overlapping primers. 0.02 pmol of pLVX_Puro digested with EcoRI and BamHI and −0.04-0.06 pmol PCR-amplified fragments were mixed with Gibson Assembly 2x Master Mix (NEB, E2611) according to manufacturer’s instructions and incubated for 1h at 50°C. 2 μL of the reaction was transformed into NEB 5-alpha Competent E. coli (NEB, C2987H) cells.
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Optimized PCR and Cloning Protocols
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