IER5 3’UTR miRNA Reporter
|2420408||1 μg, 3.0 ml, 106 cells/ml, 1.0 ml|
|Description||This lentivector, lentivirus, or stable cell line contains the 3’UTR region of the IER5 gene followed by a reporter (GFP or Luciferase). These reporters can be used to elucidate the interactions between miRNA and the 3’UTR of the IER5 gene.|
|Full Gene Name||immediate early response 5|
|Growth Medium||Use DMEM to grow stable cell lines.|
|Storage Condition||Vectors: Store at -20°C in a non-frost free freezer.|
Viruses: Store at -80°C in small aliquots and avoid repeated freeze-thaw cycles.
Stable Cell Lines: -180°C
|Titer||1 x 106 IU/ml|
|Unit quantity||1 μg, 3.0 ml, 106 cells/ml, 1.0 ml|
|Vector Map||pLenti-UTR-Luc, pLenti-UTR-GFP|
|Species||This gene is available from: Human, Mouse, Rat|
Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels.|
Grow cells in ECM-coated culture vessels with the following conditions. Freeze-Thaw Recovery:
1. Pre-warm complete media in a 37°C waterbath.
2. Remove the cryopreserved vial from the liquid nitrogen storage tank.
3. Thaw the cells quickly by placing the lower half of the vial into the 37°C water bath and remove after 60 seconds. There should still be a few ice crystals left after thawing. It is important not to over-thaw the cryovials as the presence of DMSO is toxic to the cells.
4. Re-suspend the cells in the vial and transfer the cell suspension into a 15mL sterile conical tube containing 5mL complete media.
5. Centrifuge cells at 1500rpm for 3 minutes to pellet.
6. Aspirate out the media, leaving cell pellet undisturbed.
7. Re-suspend pellet in fresh culture medium and plate in new culture vessel.
8. Incubate cultures at 37°C, 5% CO2.
Recommended Seeding Density: Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.
|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.
- Lentiviral Vector Amplification Protocol
- Selection-Drug Killing Curve
- Lentivirus Packing Protocol (for use with Vectors only)
- Lentivirus Infection Protocol
- UTR Reporters Handbook
- Enhanced Lentivirus Safety Features: Replication Incompetency
- Suggested MOI for Common Cancer Cell Lines
|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
|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.
|What is the source of the Luciferase gene?|
It is from the firefly.
|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'
|Whats the parental cell line for the 3'UTR stable cells?|
|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.
|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
|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.
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