|AX0010010||1.0 µg DNA|
This vector contains the first 417 bp of human CALCOCO2 (N-CALCOCO2) in p3XFlag-CMV-10. This vector was submitted to the abmXchange program by Huitao Liu.
|Unit quantity||1.0 µg DNA|
|Alternate Accession Numbers|
|Full Gene Name||Calcium Binding And Coiled-Coil Domain 2|
|Cloning Sites||EcoRI/ BamHI|
|Reporter/Tag||N-term 3X FLAG|
|Storage Buffer||10mM Tris-HCI, 1mM EDTA, pH8.2|
|Insert Sequence||1 atggaggaga ccatcaaaga tccccccaca tcagctgtct tgctggatca ctgtcatttc|
61 tctcaggtca tctttaacag tgtggagaag ttctacatcc ctggagggga cgtcacatgt
121 cattatacct tcacccagca tttcatccct cgtcgaaagg attggattgg catctttaga
181 gtggggtgga agacaacccg tgagtattac accttcatgt gggttacttt gcccattgac
241 ctaaacaaca aatcagctaa acagcaggaa gtccaattca aagcttacta cctgcccaag
301 gatgatgagt attaccagtt ctgctatgtg gatgaggatg gtgtggtccg gggagcaagt
361 attcctttcc aattccgtcc agaaaatgag gaagacatcc tggttgttac cactcagtga
Bacterial Beta lactamase: Ampicillin
1 year when stored at -20°C or lower in a non-frost free freezer.
|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.
|Why use a retroviral expression system?|
Expression systems based on retroviral gene delivery are generally more reliable and have broader utility than standard plasmid-based transfection systems. Retroviral gene delivery (infection) is usually used instead of transfection when cells are difficult to transfect. Retroviruses preferentially integrate into actively transcribed regions of the genome. The infection yields a cell line that stably expresses your gene of interest.
|What is the limit on the size of my DNA insert?|
Retroviruses efficiently package RNA that is approximately 8-9 kb. Most developed vectors can take as large as ~6 kb and still be packaged with no reduction in viral titer.
|Which cells are recommended to produce the retrovirus?|
Phoenix A cells are recommended for retrovirus.
|Do you have any further information about the Retro-combo mix (E-510) and the pack easy cells (E-511)?|
The Retro-combo mix is a two plasmid mixture of retro Gag/poly and VSVG. The packaging cells are a 293 derivative optimized for retrovirus production.
|I cannot detect expression for my gene of interest. Why?|
Retroviral vector transduction efficiency is very low and can only intergrate a single copy into infected target cells (for most cell types). Thus, we recommend transfection with your vector in 293 cells for a fusion gene expression assay before your viral production and infection. If your insert is in frame, the sequence is correct and there is still no signal after transfection in 293 cells, there may be technical difficulties with the western blot. Make sure the HA or His antibody is of high quality. Our Tag antibodies are carefully produced to handle most fusion vectors that are not detectable with Tag antibodies from other sources. We also have HA and His cell lysates for a true positive control. It is important to note that HA or His tag antigenecity may be altered by adjacent amino acids close to the HA or His tag. This is why some companies specify that you need a specific amino acid upstream or downstream of HA or His tags. (For example, adding an Ala before the His tag). Based on our years of experience with Tag antibodies, there are about 10% of vectors with correct fusion genes that are not detectable even by excellent antibodies.
|What is the reason for putting the SV40 promoter region after the multiclonal site?|
For retroviral vector, 5'-LTR has been traditionally used as the default promoter which will also override any down-stream promoters. If you visit other suppliers’ websites, there are quite a few retroviral vectors using 5'-LTR promoter. Later studies show that the CMV promoter down stream of 5'-LTR does not work. So both promoters are fine. Promoter is less of an issue here.
|Can I freeze the viral supernatant and use it later?|
Yes. Collect the virus at 48 hours after transfection, filter with a 0.45µm cellulose acetate or polysulfonic filter, and then freeze at –80°C. You do not need to add a cryopreservative such as glycerol or DMSO. When you are ready to use the frozen supernatant, thaw it quickly by placing it in a 37°C water bath until ice crystals have just disappeared.
|Is the poly A + sequence needed when cloning my gene of interest into a retroviral vector?|
You do not need to include the poly A + sequence with your gene because the native polyadenylation signal (from the wild-type virus) that is located in the 3' LTR of most retroviral vectors is sufficient for polyadenylation of the transcribed gene of interest.
|What is the safety concerns associated with using retroviral vectors and packaging cells?|
The National Institute of Health and the Center for Disease Control have designated retroviral vectors such as those from Moloney murine leukemia virus (MoMuLV) as Biosafety Level 2. NIH guidelines require that retroviral production and infection be performed in a Biosafety Level 2 facility. Recombinant retroviruses are extremely labile and are inactivated by ethanol, detergents, or bleach Because of their lipid-derived membrane.
|What is the difference between Retro-, Lenti-, and Adeno- viruses?|
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.
|What are the correct concentration units for each recombinant viral particle?|
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.
|How long after transduction can the infection efficiency be observed?|
You can observe transduction efficiency from 48 hours up to 5 days after infection.
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