RBM10-EGFP was generated by amplifyingRBM10from pFRT-TO-RBM1026, followed by double digestion of the PCR Product withNheI andEcoRI (NEB) and ligation intoNheI/EcoRI linearized pEGFP-N3 vector (Clontech) using T4
Aberrant translation causes ribosome stalling, which leads to the ubiquitination of ribosomal proteins and induces ribosome-associated quality control. As part of this quality control process, the E3 ubiquitin ligase RNF10 monoubiquitinates ribosomal pro
DNA was released from chromatin by adding 5 μL of cleanup buffer (900mM NaCl, 300 mM EDTA, 1.1% SDS, 4.4 mg/mL Proteinase K (NEB)) followed by an incubation for 30 min at 40°C. Tagmentated DNA was isolated using 2 × volumes of SPRI beads and eluted in 21 μl. For library...
The vector and resulting PCR product were both digested with XhoI and BamHI (NEB), and the digested products were gel purified with the QIAquick Gel Extraction Kit (QIAGEN) and ligated with T4 DNA Ligase (Promega) overnight at 4°C. Resulting clones were verified by colony PCR with QIAGEN...
Aberrant translation causes ribosome stalling, which leads to the ubiquitination of ribosomal proteins and induces ribosome-associated quality control. As part of this quality control process, the E3 ubiquitin ligase RNF10 monoubiquitinates ribosomal pro
O-Fucosylation plays crucial roles in various essential biological events. Alongside the well-established O-fucosylation of epidermal growth factor-like repeats by protein O-fucosyltransferase 1 (POFUT1) and thrombospondin type 1 repeats by POFUT2, we re
insertions can be created by incorporating half of the desired insertion into the 5′ ends of both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.Figure 4: NEB’s Q5 SDM Kit delivers higher transformation efficiency than Agilent’s QuikChange...
T4 DNA Ligase. Replacement of the inducible copy of the mutant MjYRS gene with the AzF mutant was confirmed by DNA sequencing which yielded plasmid pEVOL-BoFRS/AzFRS. To replace the constitutive copy of the mutant MjYRS pBK-AzFRS and pEVOL-BoFRS/AzFRS were treated with NdeI and PstI ...
buffer (2.5 times volume of cell pellet, 20 mM Tris-HCl (pH7.4), 20 mM KCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, 1 mM DTT, 1 × proteinase inhibitor cocktail and 40 U/mL RNase Inhibitor). And then high salt buffer (0.5 times volume of ...
To obtain the shAsterix line, the short hairpin sequence was ligated into the pValium20 vector (Ni et al., 2011) using T4 DNA ligase from NEB (M0202), according to the manual, and then integrated into the attP2 landing site (BDSC #25710). Hairpin sequences are listed in Key ...