cDNA encoding mmu-miR-302-367 (chr3: 127,545,078–127,545,959) was inserted between the BamHI and NotI sites of pCX4pur,56 (link) yielding pCX4-302-367. 293T cells were transfected with pCX4-302-367, pVPack-GP (Agilent), and pVPack-Ampho (Agilent) using FuGENE HD reagent (Promega). The culture supernatant was recovered after incubation at 37°C for 3 days and then filtered through a 0.45-μm cellulose acetate filter. Viral copy number was measured using a Retroviral Titer Set (Takara Bio), and HCT116 cells were infected with 1 × 109 copies of virus supplemented with 4 μg/mL polybrene. 3 days after infection, the cells were treated with 0.2 μg/mL puromycin to select cells that stably express mmu-miR-302-367. Provirus copy number was measured based on the extracted genomic DNA using a Provirus Copy Number Detection Primer Set (Takara Bio) following the manufacturer’s instructions.
Provirus copy number detection primer set
Manufactured by Takara Bio
The Provirus Copy Number Detection Primer Set is a laboratory tool used to quantify the number of copies of a provirus present in a sample. The primer set is designed to detect and amplify specific DNA sequences, allowing for the accurate measurement of proviral load. This product is intended for research use only and its performance characteristics have been validated by the manufacturer.
Automatically generated - may contain errors
2 protocols using provirus copy number detection primer set
1
Establishing Stable mmu-miR-302-367 Expression
2
Quantifying Proviral Load in TCR Transduced T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNAs were isolated from patient’s blood using the DNA extraction kit (Qiagen). Primers for proviral DNA sequence (retroviral packaging signal region, existing in TCR transduced cells) and human IFNg DNA (genes of whole T cells) were from Provirus Copy Number Detection Primer Set (6167, Takara Bio Inc.). qPCR assay were performed using Cycleave PCR Core Kit (CY501, Takara Bio Inc.). The DNA samples were amplified by 50 cycles of 3-step PCR reactions. Serially diluted DNA control template for provirus and IFNg were amplified and the standard curve was generated. Each DNA concentration of IFNg or proviral vector for MAGE-A4 si-TCR expression was calculated from the standard curve. The copy number was represented as the ratio of proviral DNA and IFNg DNA values.
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!