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T4 pnk m0201s

Manufactured by New England Biolabs

T4 PNK (M0201S) is a recombinant T4 polynucleotide kinase enzyme that catalyzes the transfer of a phosphate group from ATP to the 5' hydroxyl terminus of DNA, RNA, or oligonucleotides, enabling their subsequent use in various molecular biology applications.

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3 protocols using t4 pnk m0201s

1

Conditional EZH2 Knockdown with Tet-inducible System

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tet-pLKO-shEZH2 was constructed by inserting the EZH2 shRNA (the sense sequence is 5′-TATTGCCTTCTCACCAGCTGC -3′) into the tet-pLKO-puro vector (Addgene) digested with AgeI and EcoRI restriction enzymes (NEB) and dephosphorylated for 30 min at 37 °C. The digested plasmid was run on a 1% agarose ge l, cut out, and purified using the Wizard SV Gel and PCR Clean Up kit (Promega). The oligonucleotides were phosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (New England Biolabs, Inc.). Samples were annealed in a thermocycler at 37 °C for 30 min and then at 95 °C for 5 min and finally were ramped down to 25 °C at 5 °C per min. Annealed oligonucleotides were diluted 1:200 in RNase/DNase-free water. Ligation of the annealed oligonucleotide and digested tet-pLKO-puro plasmid was performed using Quick Ligase (New England Biolabs, Inc.). To induce EZH2 knockdown, cells infected with tet-inducible shEZH2 virus were treated with 100 ng/ml of Doxycycline (Sigma).
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2

Constructing pLentiCRISPR-CARM1 Plasmid

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pLentiCRISPR-CARM1 was constructed by inserting the CARM1 guide RNA (gRNA; 5′-AGCACGGAAAATCTACGCGG-3′). pLentiCRISPR v2 (Addgene) was digested and dephosphorylated with BsmBI restriction enzyme (Fermentas) for 30 min at 37 °C. The digested plasmid was run on a 1% agarose gel, cut out, and purified using the Wizard SV Gel and PCR Clean Up kit (Promega). The oligonucleotides were phosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (New England Biolabs, Inc.). Samples were annealed in a thermocycler at 37 °C for 30 min and then at 95 °C for 5 min and then were ramped down to 25 °C at 5 °C/min. Annealed oligonucleotides were diluted 1:20 0 in RNase/DNase-free water. Ligation of the annealed oligonucleotide and digested pLentiCRISPR v2 plasmid was performed using Quick Ligase (New England Biolabs, Inc.).
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3

Lentiviral CRISPR Knockout of CARM1

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pLentiCRISPR v2 (RRID: Addgene_52961) was used to generate the construct to knock out CARM1 as published previously (5 (link)). Briefly, pLentiCRISPR v2 (RRID: Addgene_52961) was digested with restriction enzyme BsmBI (NEB), then separated in the agarose gel (1%). The correct DNA band in agarose gel was cut and purified by QIAquick gel extraction kit (QIAGEN, catalog no.166047244). gDNA oligo pairs were phosphorylated by T4 PNK M0201S; NEB) in T4 ligation buffer (NEB) and annealed in a thermocycler with slow temperature ramp down from 95°C to room temperature at 5°C/minute. The ligation between the digested pLentiCRISPR v2 plasmid and the annealed gDNA (diluted 1:200 in RNase/Dnase-free water) was performed using Quick Ligase (NEB). Human CARM1 gRNA (5′-AGCACGGAAAATCTACGCGG-3′) and mouse Carm1 gRNA (5′-TCGCGTCGCCGATAGTGAGG-3′) were used for cloning.
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