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Product Details
| Cat # +Size | K1471-100 |
|---|---|
| Size | 100 Rxns |
| Kit Summary | An ideal Lentivirus Quantification Kit based on qPCR |
| Detection Method | qPCR based quantification kit |
| Applications | An ideal tool to quantify Lentivirus |
| Features & Benefits | • Reliable and Ready-to-use
• Minimal non-specific background • High specificity and sensitivity • Results ready in less than 1 h |
| Kit Components | • 2X qPCR MasterMix • Primer Mix • Standard Control DNA • ROX Reference Dye • Nuclease-Free Water |
| Storage Conditions | -20°C |
| Shipping Conditions | Dry Ice |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
BioVision’s Lentivirus qPCR Quantification Kit is a one-step assay by qPCR which does not require additional lysis or reverse transcription (RT) steps, and can be completed under one hour. Our 2X qPCR MasterMix has intrinsic reverse transcriptase activity, thus eliminating the need for an extra reverse transcription incubation step. The kit is designed to deliver high sensitivity and ensures minimal non-specific background. ROX reference dye is provided separately from the MasterMix, making this kit universally compatible with most qPCR instruments.
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How is the virus lysed?
The virus is denatured during the initial heating step of the qPCR (i.e., Enzyme Activation, 95°C).
How is the viral RNA amplified, since there is no RT incubation step?
2X qPCR MasterMix has intrinsic RNA-dependent DNA polymerase activity (reverse transcriptase activity), thus eliminating the need to add RTase or an extra RT incubation step.
Which part of the viral sequence is targeted by K1471?
The primers in this kit target the 5'LTR region.
Why do I get higher titers for the more diluted sample from the same stock?
This means that the virus stock may have strong PCR inhibitors in the sample. Thus, it would best to trust the more diluted samples results, as it lessens the inhibitor's influence.
Will the transfected plasmids used to produce the lentiviral particles interfere with the qPCR results? What about the host's genome?
The transfection plasmids and hosts genome remain in the packaging cells while the viral particles are harvested from the supernatant. The titering process will not involve the cells. Thus, the plasmids and genome will not interfere with the results as well.
My experiment needs Multiplicity of Infection (MOI), 10. How can I know that viral particle? How to convert IU (infectious units)/ml convert to viral particle?
Multiplicity of Infection (MOI) = (Product Titer (IU/ml) x Virus Volume (ml)) / Total Cell Number
For example, to achieve a MOI of 10 with a 1 x 10^6 IU/ml titer virus on 1 x 10^5 cells, use 1.0 ml of the virus.
For example, to achieve a MOI of 10 with a 1 x 10^6 IU/ml titer virus on 1 x 10^5 cells, use 1.0 ml of the virus.
Does your titration kit work with pLKO.1 based siRNA lentivirus?
Our titer kit should be able to detect pLKO.1 vector with an qPCR product size of about 100 bp.
Is this kit suitable for titrating retroviruses and adenoviruses?
No, this kit is designed for Lentiviruses only.
The dissociation curve shows that the Tm of my Lentivirus is lower than that of standards, why?
The Tm of a qPCR product is mainly depending on two variables, the chemical compositions of the reaction mixture and the sequence of the amplicon. Since all the reactions in this case have almost the same compositions (i.e. all the input templates are basically water, not for instant high salt template solution), the main variation would be the template sequence. It is possible that your the vector has a slightly different sequence (likely having less GC content) than our Standards sequence. Thus, having a lower Tm than the Standard.
Will the qPCR Lentivirus Titration(Titer) Kit amplify the integrated genomic region of the lentivirus?
No, the qPCR Lentivirus Titration Kit will not amplify the integrated genomic regions of lentivirus because the primers amplify the 5' LTR of the lentivirus.
Does the assay detect free viral RNA and so overestimate the viral titer? If so, is there a way to minimize this?
In theory, it is possible that K1471 can detect free viral RNA present in the supernatant collected during lentivirus production. However this should not impact the titer to any significant extent and should be very minimal in detection, if any. If desired, you can purify your virus to remove any free viral RNA.
For lentivirus titering, is it possible to start with an ultracentrifuged lenti sample (suspended in PBS) as opposed to straight supernatant?
Yes, starting with an ultracentrifuged lentivirus sample should be suitable for use with our Lentivirus qPCR Titration Kit.
Is this kit suitable for detecting both 2nd and 3rd generation lentivirus?
Yes, the kit is suitable for detecting both 2nd and 3rd generation lentivirus.
What type of vectors will this kit work with?
The titer primers supplied in K1471are designed for all HIV-1 based viral detection.
Is DNAse treatment required after harvesting the lentivirus stock?
DNase treatment is not recommended. When packaging lentivirus, we recommend removing the transfection medium from the cells after overnight incubation, and replace with complete culture media for the cells. Incubate another 24 hours, and then collect the supernatant.
What is the primer design based on?
The primers in this kit uses a set of proprietary sequences designed based on the 5'LTR region.
How come my cells are not successfully infected with the evaluated titer from the kit?
The infection success is dependent on the cell type. Some cells may be more susceptible than others for viral infection. Thus, the same number of infectious units may perform differently on different cell types.
What is the reporter setting to use for the qPCR machine Viia™ 7?
Lentivirus qPCR Quantification Kit is SYBR based and not probed based. So, the end user does not have to choose a reporter. Please select "SYBR Green Reagents" on the qPCR machine and set the passive reference to “ROX”.
