Having obtained this sgRNA, we added 3 µL of BsaI and 3 µL of 1× CutSmart buffer (NEB) to 1 µg pRGEB32 vector and DW to a final volume of 30 µL and heated this mixture for 1 h 30 min at 37 °C. For ligation of the double-stranded sgRNA and a BsaI-cut pRGEB32...
the sample is centrifuged at 15700 gat 4 °C for 10 min. The supernatant corresponds to the nuclear protein extract and Balance Buffer (20 mM HEPES pH 7.4, 1 mM MgCl2, 10 mM KCl) is added to reach 150 mM NaCl
An aliquot of 75–85 pmol of plasmid DNA was buffer exchanged to H2O and mixed with CutSmart buffer (NEB) and water to a final volume of 200 µL. Fifty units of BsaI-HF and 50 units of DraIII-HF were added to digest for 8–10 h, then another 20 units of each enzyme was ...
For a typical reaction, either 200 units of DraIII-HF (NEB) (for recP1P2) or 200 units of BsaI-HF (NEB) (for recP4P5) or 200 units of both DraIII-HF and BsaI-HF (NEB) (for recP1 and recP5) were added to 200 pmol of plasmid DNA in 200μl 1x NEB CutSmart buffer. After ...
Amplified product was digested with HindIII and SpeI restriction endonucleases in the CutSmart buffer system (New England Biolabs, Ipswich, MA, USA) and cloned into the gel-purified pMIR-REPORT vector digested with the same enzymes. QuikChange II XL Site-Directed Mutagenesis Kit (Agilent ...
Plasmid and PCR DNA were digested with EcoRI-HF (New England Biolabs, NEB Ipswich, MA, R3101) and KpnI-HF (NEB, R3142) in cutsmart buffer (NEB, B7204) at 37 °C according to manufacturer protocols. Plasmid DNA was dephosphorylated with calf intestine phosphatase (CIP, NEB, M0290) at...
N6-methyladenosine (m6A) mRNA modification is essential for mammalian and plant viability. The U6 m6A methyltransferases in other species regulate S-adenosylmethionine (SAM) homeostasis through installing m6A in pre-mRNAs of SAM synthetases. However, U6
Samples were treated with CIP or T4 PNK by addition of “Cutsmart” or “PNK” buffer from New England Biolabs and 10 units of CIP or T4 PNK and incubation at 37 °C for 15 min. Mock treated samples contained only Cutsmart buffer and water in lieu of CIP or T4 PNK. ...
PCR products were digested at 37°C for 3 hr in the presence of NEBuffer Cutsmart (NEB Inc, cat. No. B7204S) and BSA (NEB Inc, B9001). Digested PCR products were purified by PCR purification kit following manufacturer instruction). Ligation was performed (3:1, insert:vector molar ...
Samples were re-resuspended in 500 μL 1.2 × CutSmart restriction digest buffer (NEB). 20% SDS was added to a final concentration of 0.3%, and samples were incubated for 1 hr at 37°C while shaking at 900 rpm on a thermomixer. 20% Triton X-100 was added to a final concentration ...