Hsa-miR-22 overexpression plasmid (pGCMV/EGFP) and the negative control (NC, empty pGCMV/EGFP plasmid) were supplied by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequence of the miR-22 used within the plasmid is 5′-ACGGUACCCCGGCUGGGUGUU-3, the scrambled sequence was used within the NC group is 5′-ACGGUACCCCGGCUAGGGUGUC-3. The human S100A11 gene was constructed into a pcDNA3.1+HA empty vector (Invitrogen; Thermo Fisher Scientific, Inc.) and the NC of the pcDNA3.1+HA-S100A11 group was the pcDNA3.1+HA-empty group. Untransfected MG-63 cells were employed as a blank control group. For transfection, MG-63 cells were seeded in a 24-well plate at a density of 1×105/ml for 24 h and were subsequently transfected with 100 nM plasmids using Lipofectamine® 2000 (DNA/Lipofectamine® 2000=1/2.5; Invitrogen; Thermo Fisher Scientific, Inc.) and incubated for 6 h at 37°C. After 6 h post-transfection, the medium was changed to 2% FBS-DMEM with blasticidin (12 µg/ml) for 15 days at 37°C. Cells where stable transfection was verified were stored in liquid nitrogen for further experiments. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to determine stable transfection as described below.
Pcdna3.1 ha empty vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The pcDNA3.1+HA empty vector is a plasmid DNA vector commonly used in molecular biology experiments. It serves as a backbone for cloning and expressing recombinant proteins in mammalian cell lines. The vector contains key elements such as a cytomegalovirus (CMV) promoter, a polylinker region for inserting genes of interest, and an HA (hemagglutinin) epitope tag sequence.
Automatically generated - may contain errors
2 protocols using pcdna3.1 ha empty vector
1
Overexpression of hsa-miR-22 in MG-63 cells
2
Regulation of p63 expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected at 60–80% of confluency. For silencing experiments Lipofectamine-RNAiMAX (Invitrogen, USA) was used, siRNA#2 for p63 (Sigma, St. Louis, MO, USA) and scramble as a control (Sigma, lot. WDAA1199), RNAimax/siRNA (2:1), according to manufacturer’s protocol. Cells were harvested after 72 h.
For overexpression experiments, 300,000/dish 60 mm plated cells were transfected used Effectene Transfection reagent (Qiagen, Germany) pCDNA 3.1-HA-T∆p63γ vector and pCDNA 3.1 HA empty vector (Invitrogen, Waltham, MA, USA) as a control, according to manufacturer’s protocol (2 µg plasmid/25 µL effectene reagent). Cells were harvested after 24 h.
For overexpression experiments, 300,000/dish 60 mm plated cells were transfected used Effectene Transfection reagent (Qiagen, Germany) pCDNA 3.1-HA-T∆p63γ vector and pCDNA 3.1 HA empty vector (Invitrogen, Waltham, MA, USA) as a control, according to manufacturer’s protocol (2 µg plasmid/25 µL effectene reagent). Cells were harvested after 24 h.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!