Blank (Control) Dual Luciferase Stable Cell Line

CAT.NOUNITPRICE
m0331x106 cells / 1.0 ml
$1,350.00

Specifications


Description

Control stable 293 cell line expressing firefly luciferase and renilla luciferase.

SKUm033
SpeciesHuman (H. sapiens)
Growth PropertiesAdherent
Seeding DensityThaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.
ReporterLuciferase
SystemStable Cell Line
Mammalian Selection MarkerPuromycin
Unit quantity1x106 cells / 1.0 ml
Propagation Requirements

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.

Subculture Protocol
  1. Subculture when the culture reaches 80-85% confluency or above.
  2. Warm complete medium, Trypsin-EDTA (TM050) solution, and DPBS (Ca++ and Mg++ free) to room temperature.
  3. Rinse the cells with DPBS.
  4. Add 2-3 ml of Trypsin-EDTA (TM050) solution into flask. Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the flask in a 37°C incubator for 1 to 2 minutes or until cells completely round up. Use a microscope to monitor the change in cell morphology.
  5. At the end of incubation, gently tap the side of the flask to dislodge cells from the surface. Check under a microscope to make sure that all cells detach.
  6. Add 6-8 ml of complete medium to the flask and transfer detached cells to the 15 ml centrifuge tube. Rinse the flask with another 3 ml of complete medium to collect the residual cells.
  7. Examine the flask under a microscope for a successful cell harvest by looking at the number of cells being left behind; there should be less than 5%.
  8. Centrifuge the 15 ml centrifuge tube at 200x g for 2-3 minutes. Resuspend cells in fresh complete culture medium.
  9. Count and plate cells in a new prepared culture vessel.
Storage Condition

Storage Temperature: -180°C

Disclaimer1) 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 TypeReporter Cells
FAQs


I am very interested in your miRNA products. How about the price for the 3' UTR Luciferase/GFP Reporters? Could I directly order with your company or online and pay by PO?
To search for the price of 3'UTR product, please go to http://www.abmgood.com/microRNA-miRNA/targetvalid.php?csn=26&ssn=11838&dsn=12288, and enter the gene name. You can place your order by email, telephone, fax, mail or online. PO number or Visa can be used. Please see the detail by clicking the following link http://www.abmgood.com/misc/orders.php
How does the 3'UTR platform work?
The stable cell line or the infected cells from the lentivirus will consistently express luciferase/GFP unless there are miRNA that activate the 3'UTR to "knockdown" the reporter expression.
What is the forward and reverse sequencing primers for the 3'UTR luciferase reporter systems?
Forward sequencing primer: Luc Forward primer 5'-GCAAGTTGGACGCCCGCAAGATC-3' Reverse sequencing primer: SV40 promoter reverse primer 5`-TAGTCAGCCATGGGGCGGAGA -3'
What is the upper size limit for the 3'UTR insert for viral packaging?
For GFP, we can fit a 3.5kb insert for efficient packaging. For Luc, we can fit a 2.6kb insert for efficient packaging. The above size limits can be stretched a little (~0.5kb-1kb larger), but the packaging efficiency and virus titer will be lower.
Could 3'UTR GFP reporter be used with a conventional transient transfection system (e.s.Lipofectamin 2000)? Could this vector be used to validate the activity of an endogenous overexpressed miRNA?
Yes, this vector can also be transiently transfected using a transfection reagent such as lipofectamine 2000, or DNAfectin available from abm. This reporter construct can also be used to validate interactions of an endogenously expressed miRNA, if the known target site is present in the 3' UTR sequence.
References


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