0610007L01Rik ORF Vector
|1000201||1.0 µg DNA|
|Description||The 0610007L01Rik ORF Vector holds the gene (cloned by a restriction enzyme-independent method) between an AflII and EcoRV cut site. Inserts are flanked by and can be excised using AflII and EcoRV as long as inserts do not contain any internal AflII and EcoRV sites. In the case that the insert contains these internal cut sites, the end user can PCR amplify the insert with preferred restriction sites for subcloning.|
|Aliases||A930023A16Rik; AW557951; G430067H08Rik|
|Unit quantity||1.0 µg DNA|
|Storage Buffer||10mM Tris-HCI, 1mM EDTA, pH8.0|
|Species||This gene is available from: Mouse|
|List of bacterial markers||Spectinomycin|
|Guarantee||abm guarantees that the correct ORF construct is provided. If this is not the case, we will provide a one-time replacement. The replacement will not be covered by the same guarantee.|
|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.
|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.
|For the ORF plasmids, it states the following in the description: "The last nucleotide of the stop codon has been modified, allowing for subsequent generation of C-terminal fused proteins." How has the stop codon been modified? How can the modified stop codon be used?|
The last nucleotide change of stop codon actually removes the stop codon (for example from TGA to TGC). Thus, the translation will not stop there but stop at the next available stop codon. All the ORF cDNA in our library do not have the stop codon. The removal of the stop codon will facilitate the expression of C terminal fusion protein. To use this ORF plasmid, the customer can use PCR to introduce the RE cutting sites. The customer can introduce the stop TGA at this step at the same time. If the customer wants to use the recombinant cloning way, then the customer needs to make sure the vector has its own stop codon, either right after the inserted gene or after the C terminal tag sequence.
|Why is the provided vector sequence so much longer than the vector map size?|
The sequence contains a 2443bp region between the AfIII and EcoRV cut site, which would be removed during the cloning process of the insert. AflII occurs at 554bp-560bp and EcoRV occurs at 2998bp-3003bp.
|Why is the vector sequence online at 5kb but the vector map states a 2.6kb size?|
The vector sequence contains the death gene that gets removed upon recombination with the ORF insert. The vector map is the more accurate depiction of what the vector will be with the ORF inserted.
|Are any other cut sites available?|
HpaI and XbaI are also useable to cut out the insert CDS from the vector, besides the stated AflII and EcoRV.
|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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