Briefly, cells were lysed in RIP lysis buffer and incubated with METTL13 antibody or IgG overnight at 4 °C. After washing with PBS, the RNAs co-precipitated by METTL13 were extracted and analyzed using real-time PCR. To explore the binding proteins with METTL13, a Co-IP assay was ...
The target fragments were inserted into the pMIR-reporter plasmid after restriction endonuclease digestion by T4 DNA ligase. The correctly sequenced luciferase reporter plasmids WT and mutant (MUT) were co-transfected into HEK-293 T cells (Shanghai Beino Biotechnology Co., Ltd., China) with miR-...
The beads were then washed by 1xTBS buffer (50 mM Tris–HCl, pH=7.4, 150 mM NaCl) three times after removing the supernatant. The washed beads were re-suspended in 50 µL 1xSDS-PAGE sample buffer and boiled at 100°C for 5 mins. 10µL protein sample was used for Western blot...
Constructs. RBM10-EGFP was generated by amplifying RBM10 from pFRT-TO-RBM1026, followed by dou- ble digestion of the PCR Product with NheI and EcoRI (NEB) and ligation into NheI/EcoRI linearized pEGFP-N3 vector (Clontech) using T4 DNA ligase (NEB). The tet-on lentiviral plasmids ...
This vector DNA is designated V2. [0146] Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBacHDGNR10) with the HDGNR10 gene using the enzyme BamHI. The sequence ...
(Gene ID: 101928483) were designed by an online tool (http://www.e-crisp.org/E-CRISP/designcrispr.html). The coupled complementary DNA oligos were annealed and inserted into the BsmBI sites of linearized LentiCRISPR v2 vector using T4 DNA ligase. The sequences of sgRNA NALT1 were 5′-...
In brief, cells or tissue pieces to be genotyped were lysed by being boiled in lysis solution (25 mM NaOH [pH 12] and 0.2 mM EDTA) for 20–30 min for the extraction of genomic DNA.23 Alkaline pH was neutralized by the addition of an equal volume of neutralization buffer (40 mM Tris...
Standard cloning methods are used in the construction ofPseudomonas fluorescens(Pf) expression plasmids engineered to produce full-length DIG-10 proteins encoded by plant-optimized coding regions. Restriction endonucleases are obtained from New England BioLabs (NEB; Ipswich, Mass.) and T4 DNA Ligase (...
EMSA Cells were treated with SDF-1α for the indi- cated times, rinsed with PBS, and pelleted. Cyto- plasmic proteins were isolated by lysing the cell membrane using buffer containing 10 mmol⋅L-1 Hepes, pH 7.6, 60 mmol⋅L-1 KCl, 1 mmol⋅L-1 EDTA, 0...
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 DNA ligase (NEB). The tet-on lentiviral plasmids carryingRBM10-EGFP were con...