The C-terminal Myc/DDK-tagged pCMV6 empty vector and the human CD28 (NM_006139), KANSL1L (NM_152519) and USP37 (NM_020935) expression vectors were purchased from Origene. Constructs carrying mutant alleles were generated by direct mutagenesis with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies), according to the manufacturer’s instructions. The full-length wild-type and mutant human CD28 cDNAs were inserted into the V5/His-tagged pcDNA3.1 plasmid, with the directional TOPO expression kit (Thermo Fisher Scientific). HEK293T cells were transiently transfected with the various constructs, with a calcium phosphate kit (Thermo Fisher Scientific) or in the presence of X-tremeGENE 9 DNA Transfection Reagent (Roche), according to the manufacturers’ instructions.
Calcium phosphate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Calcium Phosphate Kit is a laboratory reagent used for the quantitative determination of calcium and phosphate levels in various sample types. It provides a reliable and efficient method for measuring these important analytes.
Automatically generated - may contain errors
4 protocols using calcium phosphate kit
1
Cloning and transfection of human CD28, KANSL1L, and USP37
2
SARS-CoV-2 Spike Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SARS-CoV-2 S gene (GenBank: QHU36824.1 ) fusion with a c-terminal 12xHis tag was synthesized and cloned into a pcDNA3.1 vector. Phoenix cells were grown in DMEM containing 10% FBS and co-transfected by pLV-mCherry and pcDNA-Spike or pMD2.G vector using a calcium phosphate kit (ThermoFisher, Waltham, MA, USA). The supernatant with produced virus (Spp or VSV-G lentivirus) was harvested 72-hours post transfection, clarified by centrifuging at 5000 g for 15 min followed by filtration of the supernatant through a 0.45 μm filter disk. The virus was collected by an ultracentrifugation at 24,000 rpm for 2 hours (hrs) using Beckman SW41 rotor. The viral pellets were resuspended by cold PBS buffer and stored at −80 °C before use. The viral particle number was determined using a real time RT-PCR assay to quantify the RNA copies of mCherry.
3
Purification and Analysis of Virus-Like Particles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient transfections of the expression vectors (4 micrograms (µg) of full-length Gag eukaryotic expression plasmids (FN7 and FN9) along with 2 µg of MPMV-based transfer vector SJ238 (link) were carried out in HEK 293T cells in triplicates using a calcium phosphate kit (Invitrogen) following manufacturer’s recommendations. The resulting supernatants from the transfected cultures containing virus particles were subjected to low-speed centrifugation (bench-top centrifuge, 3,700 g for 10 minutes) to clear cellular debris. Next, supernatants were filtered using 0.2 μm surfactant free cellulose acetate (SFCA) syringe filters and subjected to ultracentrifugation at 70,000 g to pellet virus like particles (VLPs) using a 20% (w/v) sucrose cushion. The pelleted VLPs were resuspended in TN buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl) and processed for RNA extraction and western blotting. RNA isolation was performed using TRIzol®.
4
Monitoring Gag Expression in Eukaryotic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of Gag in eukaryotic cells was monitored in transient transfections using calcium phosphate kit (Invitrogen, Carlsbad, CA, USA) in HEK 293T cells. The transfections were carried out in triplicates in 6-well plates using 4 micrograms (µg) of full-length Gag eukaryotic expression plasmids (AK13 or AK14) along with 2 µg of MMTV-based transfer vector, DA024 [30 (link)]. To monitor transfection efficiencies, a secreted alkaline phosphatase expression plasmid (pSEAP, at a concentration of 100 ng per well) was also included in the transfections. Approximately 72 h post transfection, supernatants from the transfected cultures were harvested and subjected to low-speed centrifugation (3700× g for 10 min) to clear cellular debris. The clarified supernatants were then filtered through 0.2 µm cellulose acetate syringe filters, and subjected to ultracentrifugation at 70,000× g with a 20% (w/v) sucrose cushion to pellet the VLPs. The pelleted VLPs were then resuspended in TN buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) and subjected to RNA extraction (TRIzol) and western blotting.
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!