Blank (Control) 3'UTR Luciferase Stable Cell Line
|m018||1x106 cells / 1.0 ml|
Stable 293 cell line expressing Luciferase from Photinus pyralis.
|Species||Human (H. sapiens)|
|Seeding Density||Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.|
|System||Stable Cell Line|
|Mammalian Selection Marker||Puromycin|
|Unit quantity||1x106 cells / 1.0 ml|
Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is recommended for optimal cell adhesion to the culture vessels. The basal medium for this cell line is Prigrow III medium available at abm Cat. No. TM003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (Cat. TM999) at a final concentration of 10%, Penicillin/Streptomycin Solution (Cat. G255) at a final concentration of 1%, and 0.6µg/ml Puromycin (Cat. G264) to maintain its transgene expression. Carbon dioxide (CO2): 5%, Temperature: 37.0°C.
Storage Temperature: -180°C
|Disclaimer||1) Disclaimer for Transcript Variants: The provided accession number refers to the transcript (mRNA) sequence for this product. The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. All sales are final.|
2) Disclaimer for Gene Sequence: The provided accession number refers to the transcript (mRNA) sequence for this product. Please verify that this is the desired transcript sequence by cross-referencing. This is important because a single gene can have multiple different transcripts owing to naturally occurring variations. All sales are final.
3) Disclaimer for Intended Use: All of abm's vectors and viral particles are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its vector(s) in therapeutic/diagnostic application(s).
4) Disclaimer for Extra Nucleotides: Cloning may lead to the insertion of extra nucleotides at the 5' or 3' end of the target sequence which, in most cases, is innocuous to the stability/functionality of the construct.
5) abm guarantees that at least 1 out of the 3 sgRNA constructs purchased in a set designed to be used with Cas9 Nuclease will result in gene knock-out due to frameshift mutations in over 50% of cells, after successful infection and drug selection. This guarantee applies to sgRNAs designed to target human, mouse or rat genes only. If knock-out is not achieved in extremely rare cases, a one-time replacement of another set of 3 targets with alternative sgRNA sequences will be provided. To qualify for this replacement, customers must examine knock-out efficiency by Surveyor assay. Before sending your inquiry, please make sure you have optimized your experiments as far as possible. This includes (where applicable) increasing and optimizing your MOI, increasing the duration of infection (up to 72 h), and carrying out clone screening before assaying for knock-out. Please also provide data to show that a reporter virus was used to optimize the MOI for your target cell line. Customers must provide adequate data to show >80% infection efficiency with a positive control, plus additional qPCR data to evaluate the level of mRNA expression.
For vector transfection, please evaluate the vector transfection efficiency by detecting Cas9 or puromycin expression for the 'All-in-One' vectors using qPCR, or neomycin for constructs containing only the sgRNA. In addition, please provide Surveyor Assay or Sanger Sequencing data on at least 20 isolated clones.
abm limits its obligation and liability for the success of this technology to providing one replacement of any sgRNA lentivector product only. The replacement set will not be covered by the same guarantee. If these constructs are also considered to be ineffective then the gene is most likely not susceptible to sgRNA knock-out.
|Cell Type||Reporter Cells|
|Is it possible to use your kit (miRNA cDNA Synthesis Kit) to do quantitative PCR with the Taqman strategy?|
This kit along with our miRNA primers would not be compatible for use with Taqman strategy.
|How does the universal reverse primer work?|
The universal reverse primer can be used for all mature miRNA. To quantify or amplify a specific miRNA, the universal reverse primer with the appropriate forward miRNA primer needs to be used. Our forward primers can work with any kits. Our universal primers will only work with abm's cDNA synthesis kit.
|Can I use SYBR green mastermix for downstream qPCR of the cDNA produced with this kit?|
It may be possible to use a SYBR green mastermix for qPCR, however, our BrightGreen miRNA miRNA qPCR MasterMix recipe has been fully optimized to provide the most successful conditions for qPCR. We have performed extensive testing with minor differences in the composition of miRNA qPCR buffer, this has proven to give very dramatic differences to the results of qPCR. General qPCR mastermixes have more additives to prevent non-specificity and formation of primer dimers and the conditions are often too harsh for the miRNA primers to anneal properly
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