Inhibitor hsa-miR-4266 AAV miRNA Vector
| Cat. No. | Amh3076500 |
| Name | Inhibitor hsa-miR-4266 AAV miRNA Vector |
| Unit | 500 ng |
| Description | This miRNA inhibitor AAV vector is part of abm’s AAV Expression System and can be packaged into virus using an appropriate packaging mix to inhibit your miRNA of interest in a wide range of host cells or animal models. This construct contains a GFP reporter for monitoring transfection or transduction efficiency. |
| miRNA Name | hsa-miR-4266 |
| Accession Number | MIMAT0016892 |
| Species | Human (H. sapiens) |
| Vector Map | pAAV-mir-OFF-hGH-amp (click blue link to view) |
| miRNA ID | MIMAT0016892 |
| System | AAV Vector |
| Vector Size | 5732 |
| Storage Condition | AAV Vectors are shelf stable for 1 year when stored at -20°C or below. Shipping and short term storage are acceptable at ambient temperature. Avoid repeated freeze-thaw cycles. |
| QC | Restriction Enzyme Digest and Sequencing |
| Material Citation | If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. Amh3076500 |
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Supporting Protocol
| How should I store my lentivirus? | |
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The lentivirus should be aliquoted into smaller working volumes and then stored at -80°C. Lentivirus is sensitive to storage temperature and to freeze/thaws. It can lose up to 5% or more activity with each freeze/thaw. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
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| Is it ready for use? | |
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Yes, it is ready to use. Just load suggested volume (5ul) to the well.
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| What is the smallest size agarose gel can separate? | |
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That is around 300bp for 1% agarose and 100bp for 2.5% agarose. With anything smaller than the dye, there may be separation but it will be difficult to tell when the fragment will run off the gel.
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| What generation does abm use for their lentiviral transfer vectors? | |
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abm’s lentiviral transfer vectors use the third generation lentivirus system. It is based on the inactivated HIV genome. Note that our lentivirus packaging plasmids cannot be integrated into the host and are transiently expressed.
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| What is the difference between Retro-, Lenti-, and Adeno- viruses? | |
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Retrovirus: Classic, can integrate into the genome but with low transduction efficiency. They are useful for gene transfer and protein expression in cells that have low transfection efficiency with other transfection reagents. Lentivirus: Can integrate into the genome with relatively high transduction efficiency and they are very useful for cells that have low transfection efficiency with other transfection reagents. No special competent cells required, as they are stable plasmids. Lentiviruses are a powerful tool for stable gene transfer to both dividing and non-dividing cells in vitro and in vivo. Adenovirus: Only work transiently (about 7 days) but have almost 100% transduction efficiency. Adenoviruses can infect a broad range of cell types with the highest efficiency and infection is not dependent on active host cell division. A second key feature is that high virus titers and high-level gene expression can be obtained in most mammalian cells.
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| What are the correct concentration units for each recombinant viral particle? | |
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For lentiviruses and retroviruses, they are measured in CFU/ml (colony-forming units per millilitre). Transduction with lentiviruses and retroviruses can cause the formation of colonies, which can be quantified for concentration. For AAV the titer is measured as genome copies per mL (GC/mL). Adenoviruses are measured as PFU/ml (plaque-forming units per millilitre). Transduction with adenoviruses will kill packaging cells, forming plaques in the process for quantification. The concentration for all three types of viruses can also be classified as IU/ml (Infectious Units/ml). Ultimately, the units refers to the viral particles and different units reflect the different assays involved.
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| What do I use to check if my cells were successfully immortalized by the SV40 agent? | |
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We have an SV40 T antibody that can be used for the western blot analysis. The catalog number is G202.
Otherwise, a qPCR primer can be designed on the SV40 gene for qPCR analysis. The sequence can be found in the link below:
http://www.abmgood.com/pLenti%20SV40-Vector-Location-Map.html
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| What are the primers to use for SV40 identification? | |
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SV40 Forward Primer Sequence
5’ ACTGAGGGGCCTGAAATGA
SV40 Reverse Primer Sequence
5’ GACTCAGGGCATGAAACAGG
These are qPCR primers and the band size is 61 bp.
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| What advantages / disadvantages exist between the Lenti-SV40, -SV40T, and SV40T+t vectors? | |
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There are simply differences in the content of all vectors due to customer demand for variety. Lenti-SV40 will contain the whole SV40 gene, -SV40T, the large T Antigen only, and -SV40T&t the large and small T antigens only.
It is up to the end user to decide which vectors will best suit their project, however we have successfully used Lenti-SV40 (whole gene) in a wide range of immortalization projects.
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| Are the Opti-DNA markers compatible with radiolabelling? | |
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Our lab has not tested the Opti-DNA markers for this particular application, but we believe it shouldn't be a problem. The Opti-DNA markers should be compatible for radio-labelling.
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| What is the accession number for the SV40? | |
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The SV40 covers the entire genome and the accession number is J02400.1. You can use this information to design primers for conventional PCR as well.
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| Is it possible to use frozen blood sample with this kit? | |
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Frozen blood samples have not yet been tested with this kit, however this should not be a problem.
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| What is the TE buffer used for? | |
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The TE buffer is optional and is included because some prefer to use TE for elution. TE buffer can be used instead of elution buffer if needed.
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| How long after transduction can the infection efficiency be observed? | |
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You can observe transduction efficiency from 48 hours up to 5 days after infection.
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| What are the primers to use for SV40T and SV40T tsA58 detection? | |
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PCR primers:
SV40T Forward Primer Sequence
5’ AGCCTGTAGAACCAAACATT 3'
SV40T Reverse Primer Sequence
5’ CTGCTGACTCTCAACATTCT 3'
The two primers should amplify the region between 3677-4468bp, giving a 792bp fragment.
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| What is the sequence of the SV40 large T antigen? | |
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This information can be accessed on this page by clicking on "pLenti-SV40-T" under vector map. The Large T antigen is at position 5079-5927.
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| For G221 and LV620, what does the 'V12' in RasV12 mean? | |
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The V12 means that amino acid # 12 is mutated from a Valine to a Glycine. Other than that, the sequence matches the coding region of HRAS perfectly (NM_005343).
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| Where is the SV40T tsA58 gene sequence? | |
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The SV40T tsA58 gene is located between 3138-5264bp, with the Alanine-to-Valine mutation at amino acid 438.
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| How should I store abm’s vectors? | |
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We recommend storing abm’s vectors at -20°C and avoiding repeated freeze/thaws as this may affect plasmid integrity and cloning efficiency.
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- Shi J, Gao X, Tian T, et al. Structural basis of Q-dependent transcription antitermination. Nat Commun. 2019;10(1):2925. Published 2019 Jul 2. doi:10.1038/s41467-019-10958-8.
- Trachman, R., & Ferré-D'Amaré, A. (2019). Tracking RNA with light: Selection, structure, and design of fluorescence turn-on RNA aptamers. Quarterly Reviews of Biophysics, 52, E8. doi:10.1017/S0033583519000064.
- Alexis Autour, Sunny C. Y. Jeng, Adam D. Cawte, Amir Abdolahzadeh, Angela Galli, Shanker S. S. Panchapakesan, David Rueda, Michael Ryckelynck & Peter J. Unrau "Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells" Nature Communications. 2018 Feb;23(9):656. DOI: 10.1038/s41467-018-02993-8.
- Panchapakesan SSS, Ferguson ML, Hayden EJ, Chen X, Hoskins AA, Unrau PJ. et al. "Ribonucleoprotein purification and characterization using RNA Mango." RNA. 2017 Oct;23(10):1592-1599. DOI: 10.1261/rna.062166.117.
- Trachman RJ 3rd, Demeshkina NA, Lau MWL, Panchapakesan SSS, Jeng SCY, Unrau PJ, Ferré-D'Amaré AR. et al. "Structural basis for high-affinity fluorophore binding and activation by RNA Mango." Nat Chem Biol. 2017 Jul;13(7):807-813. DOI: 10.1038/nchembio.2392.
- Cawte, Adam. et al. "Live Cell Imaging of Genomic Loci using Fluorescent RNA Aptamers." Biophysical Journal. 2017 Feb, Volume 112,Issue 3,69a. DOI: 10.1016/j.bpj.2016.11.416.
- Jeng SC, Chan HH, Booy EP, McKenna SA, Unrau PJ. et al. "Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers." RNA. 2016 Dec;22(12):1884-1892. Epub 2016 Oct 24. PubMed : 27777365.
- Ouellet J. et al. "RNA Fluorescence with Light-Up Aptamers." Front Chem. 2016 Jun 28;4:29. DOI: 10.3389/fchem.2016.00029. eCollection 2016. Review. PubMed : 27446908.
- Panchapakesan SS, Jeng SC, Unrau PJ. et al. "RNA complex purification using high-affinity fluorescent RNA aptamer tags." Ann N Y Acad Sci. 2015 Apr;1341:149-55. DOI: 10.1111/nyas.12663. Epub 2015 Jan 13.
- Dolgosheina EV, Jeng SC, Panchapakesan SS, Cojocaru R, Chen PS, Wilson PD, Hawkins N, Wiggins PA, Unrau PJ. et al. "RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking." ACS Chem Biol. 2014 Oct 17;9(10):2412-20. DOI: 10.1021/cb500499x. Epub 2014 Aug 21.
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