ZNF833 siRNA

CAT.NOUNITPRICE
6920609500 ng per target,1.0 μg,2 x 200 μl,5 x 200 μl,3 x 5 nmol
$85.00

Specifications


DescriptionThis ZNF833 siRNA is available in lentivirus, AAV, and oligo formats. It can be used to knockdown the ZNF833 gene in a wide range of host cells via transduction or transfection.
SKU6920609
Gene NameZNF833
Accession NumberNM_001013691
Accession NumberHuman:NM_001013691
Titer>1 x 109 GC/ml,>107 IU/ml
Conversion of ODFor siRNA oligos: 1 OD = 2.5 nmol siRNA (duplex) = 5 nmol siRNA (single-stranded) = 33 μg
Unit quantity500 ng per target,1.0 μg,2 x 200 μl,5 x 200 μl,3 x 5 nmol
Vector MappiLenti-siRNA-GFP,pAAV-siRNA-GFP
Vector SizepiLenti-siRNA-GFP: 9043bp
pAAV-siRNA-GFP: 6593bp
SpeciesThis gene is available from: Human
Guaranteeabm guarantees that at least one out of the three siRNA oligos purchased in a set will result in over 70% knockdown of gene expression (qPCR) within target cells showing >50% transfection efficiency. If this is not the case, we will provide a one-time replacement of three new constructs with alternative siRNA sequences. To qualify for this replacement, the oligos must be transfected at the appropriate amounts (see Troubleshooting section of the User Manual) and assayed at least 48 hours post-transfection. Customers must provide adequate data to show >50% transfection efficiency with a positive control, plus additional qPCR data or a western blot to evaluate the level of gene expression. The replacement set will not covered by the same guarantee, and if these constructs are also considered to be ineffective then it is most likely the gene is not susceptible to siRNA knockdown. abm limits its obligation and liability for the success of this technology to providing one replacement of any siRNA oligo product only. Before sending your inquiry, please make sure you have optimized your experiments as far as possible, this includes (where applicable) optimizing the siRNA concentration and the duration of transfection (up to 72 hours). Please send all replacement inquiries and experimental data to [email protected]
CautionThis product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information.
FAQs


Is there a EF1 vector with GFP for cloning gene of interest
yes, we do have the vector and I will ask our scientist to email about the vector later.
Q. About the negative control (scramble), what is it? How do you know it is not targeting other genes? Also does it have any marker for me to say that the cells have got the control siRNA, but it is not bringing down my gene?
It is specifically designed that it should bring down any gene based on sequence search. Using neomycin as your selection marker can show whether the negative control was successfully incorporated by the cell.
How come I cannot see EGFP in my cells after infection with the iLenti-EGFP product? I have seen fluorescent activity with 293T cells but no fluorescent activity in my target cells (not 293T cells).
It is easy to see EGFP in 293 cells. Most of the other cell lines take about 48-73h to see the EGFP, and even then, the fluorescence activity may be low. The promoter strength for the EGFP gene varies with different cell line. Refer to the paper "Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in a Promoter-Dependent Manner" by Xia et al. in Stem Cells and Development 16:167-176 (2007). EGFP expression is dependent on promoter strength, which may differ between different cell types. The promoter for the EGFP gene is from CMV. Even though the promoter is ubiquitous, the expression of EGFP may be suppressed in some target cells. That does not necessarily mean that the vector is not in the cells. The vector may be there and the siRNA may be expressed because the promoters for the siRNA are different. You can try Western blotting against the target gene product along with a standard curve to evaluate whether siRNA is being expressed and functional in the target cells.
About the negative control (scramble), what is it? How do you know it is not targeting other genes? Also does it have any marker for me to say that the cells have got the control siRNA, but it is not bringing down my gene?
It is specifically designed that it should bring down any gene based on sequence search. Using antibiotics as your selection marker can show whether the negative control was successfully incorporated by the cell.
How come I cannot see EGFP in my cells after infection with the iLenti-EGFP product? I have seen fluorescent activity with 293T cells but no fluorescent activity in my target cells (not 293T cells).
It is easy to see EGFP in 293 cells. Most of the other cell lines take about 48-73h to see the EGFP, and even then, the fluorescence activity may be low. The promoter strength for the EGFP gene varies with different cell line. Refer to the paper "Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in a Promoter-Dependent Manner" by Xia et al. in Stem Cells and Development 16:167-176 (2007). EGFP expression is dependent on promoter strength, which may differ between different cell types. The promoter for the EGFP gene is from CMV. Even though the promoter is ubiquitous, the expression of EGFP may be suppressed in some target cells. That does not necessarily mean that the vector is not in the cells. The vector may be there and the siRNA may be expressed because the promoters for the siRNA are different. You can try Western blotting against the target gene product along with a standard curve to evaluate whether siRNA is being expressed and functional in the target cells.
What generation is our iLenti-system?
It is third generation lentivirus.
What is our lentivirus based on?
Our system is based on inactivated HIV. All our packaging plasmids cannot be integrated into the host and they are transiently expressed only.
What is the function of 3’ acceptor sites in lentiviral vectors?
It's for biosafety reason. The 5' splice donor and 3' acceptors sites enhance the biosafety of the vector by facilitating removal of the packaging sequence and RRE such that expression of the gene of interest in the transduced host cell is no longer Rev-dependent (Dull et al., 1998). It will inhibit viral production in stable cell lines.
How do you ensure that your lentivirus is replication-defective?
We cannot send a protocol for you to view but I can summarize what we do to ensure the virus' replication deficiency. Lentiviral vector stocks are tested for the presence of replication-competent lentivirus (RCL) by monitoring p24 antigen expression in the culture medium of transduced 293T cells for 30 days. Serial passaging of the transduced p24 cells over this period allows for the amplification of RCL. Since the vectors are replication-defective, no amplification of p24 signal would normally be expected. However, if there were an increase in P24 signal over time, it would be indicative of RCL contamination. The p24 antigen is assayed with a commercial kit from http://www.zeptometrix.com/ . This assay has been performed on multiple vector lots without a positive result.
How efficient is the lentivirus transduction?
This depends on the cell line or type as each one is different. In general, the lentivirus is very efficient in most cell lines with 100% for 293 and other commonly used cells. The lowest efficiency observed is 10% and customers have to do evolution for a specific cell line using EGFP lentivirus. For customers that just need to generate a stable cell line, 1% trandsuction efficiency is enough as every transduced cell is permanently integrated with the vector. Therefore, 10ml of 10^6 cfu/ml would contain more than enough transduced cells.
Can I use this virus to infect cancer stem cell?
Theoretically, the lentivirus can transfect cancer stem cells. It is important to note that the transfection efficiency will vary with different cell types.
Do you have the scramble shRNA lentivirus vector for mouse and rat as control?
Our scramble negative control can be used for human, mouse and rat. Please refer to Cat.# LV015-G for ordering information.
Can this vector be used with the 3rd generation packaging system
Yes, our siRNA lentivectors are 3rd generation, but can be packaged using either 2nd or 3rd generation packaging systems.
Could I transiently transfect these vectors to my target cell using lipofectamine or fugene directly? If I can do in this way, how many siRNA do I need to order for one target gene. I notice for each gene you have four options, and also have ***_set. Is one siRNA lentivector enough for knock-down assay?
You can use Lipofectamine to transiently transfect the vectors to your target cells. Since the effect of an siRNA on a gene in a particular cell line can vary, we suggest choosing the set of 4 siRNA constructs to increase your chances of getting good knockdown of your target gene in your cells.
Is the siRNA set composed of lentiviral vectors with shRNAs or siRNAs? Do you validate the RNA interference?
Our RNA interference lentiviral vectors contain siRNAs. We employ a dual convergent promoter system where the sense and antisense strands of the siRNA are expressed by two different promoters rather than in a hairpin loop - to avoid any possible recombination events that can occur. In cases where there are no verified and published siRNA sequences for your gene of interest, we use our siRNA design software to locate suitable target sites. If these designed siRNAs don't give efficient knock down of gene expression in your experiments, we offer a onetime replacement (free of charge) that will contain a new set of sequences to try. ABM guarantees that at least one out of the four siRNA Lentivector constructs purchased in a set will give over 70% knockdown efficiency within appropriate target cells showing >80% transfection efficiency. If these four constructs are still considered to be ineffective, then it is most likely the gene is not susceptible to siRNA knockdown.
Can I use phoenix 293 cells to produce the lentivirus with the lentiviral siRNA plasmids you sell?
The Phoenix 293T cells are suitable for retrovirus packaging but not for lentivirus packaging. To package our lentivectors, you will need to use a 2nd or 3rd generation packaging mix, like the ones we have here: http://www.abmgood.com/Lentivirus-Packaging-Systems.html
I want to transfect the siRNA using Lipofectamine. How much of the siRNA do you recommend to transfect in a 60mm dish for efficient knock down of the gene?
Since every gene is different, it is best to optimize the transfection condition to allow for the most siRNA vector used without having toxicity. Try transfecting a series of 5ug, 8ug, 10ug and 12ug of the siRNA with your cells and the best one will be the highest siRNA used without seeing toxicity.
Are they high copy or low copy plasmids? What is expected plasmid DNA yield?
These are high copy plasmids and should be propagated with an authentic DH5a strain. We typically see a yield of 300-500ug DNA from a 250mL culture.
Is there a provided protocol I can reference when using this product?
You can find the User Manual and Infection Protocol at the following link: http://www.abmgood.com/RNAi-shRNA-siRNA-Expression-Vector.html
What is the maximum size of the insert that can be cloned into Lenti-III-EF1α Vector (cat No LV043)?
The maximum insert size for LV043 is around 4kb, above this upper limit little to no virus will be packaged.
What primers can I use to sequence my vectors?
One forward sequencing reaction using the U6 primer should be sufficient: U6 forward sequencing primer 5'-TACGTCCAAGGTCGGGCAGGAAGA-3'
What enzymes can I use to cut the plasmids for verification?
BamHI will cut out GFP in these vectors, so you should see a 0.8kb band when you cut the plasmids with this enzyme (applies only to vectors containing GFP). Another method you can use to check for the insert is to perform an EcoRI digest. The parental vector contains 2 EcoRI sites, one between the two BbsI sites which were used to clone in the insert, and one located immediately after GFP. If the insert is cloned in, the first EcoRI site will be removed, and when this is cut by EcoRI, you will only get a linearized vector band. Ones that are still the parental plasmid with no insert cloned in will still contain both EcoRI sites, and cutting with EcoRI will give 2 bands, ~8.2kb + 0.8kb. Alternatively KpnI should give a 0.9kb band if the insert is present, and a linear vector band only if there is no insert present. The siRNA insert is cloned in with a KpnI site for screening purposes.
Do you stock a non-targeting (control) siRNA lentiviral vector?
We have a scramble siRNA negative control construct (cat#LV015 without GFP, cat#LV015-G with GFP).
Does the final siRNA produced by this system have overhangs or not?
Once expressed in the host cell, Dicer will process the expressed dsRNA duplex into siRNA with 3' overhangs.
How much virus can I produce with these kits (e.g. LV301)?
LV301 will allow you to produce virus from 5-6 x 10cm pates. You will be able to amplify the vectors (and 293T cells) as much as needed, so these kits will only limit production with the amount of packaging mix and Lentifectin transfection reagent provided. These can all be purchased individually if a larger volume is required - please refer to protocol for more information.
How many ug DNA do you recommend to transfect cells in a 6 well plate?
We recommend transfecting 2ug DNA (0.5ug/ul) per well of a 6-well plate
I want to know the shRNA lentivector set contains a mix of 4 plasmids or 4 individual plasmid?
The vector sets are supplied as 4 individual constructs, only the pooled lentivirus is supplied as a mixed solution. Please also note that our system utilizes the expression of mature siRNA sequences from two convergent promoters and this is not a hairpin loop, shRNA based system.
Can you send a small aliquot of the plasmid along with the packaged siRNA virus for verification purposes?
When siRNA virus and its DNA vector counterpart are requested in the same order, we will be happy to include the unpackaged plasmid at a discounted rate - 50% off the listed vector DNA price for INDIVIDUAL constructs and 40% off listed vector DNA price for SETS OF 4. This discount must be requested at the time of ordering and cannot be applied to subsequent orders placed after shipment of the virus.
Can you please advice on a protocol for vector extraction from filter paper?
1) Cut out the circle on the filter paper (this is where the DNA will be spotted), and then cut it into tiny pieces into a 1.5ml tube. 2) Put 30-50ul of PCR-grade water, 10mM Tris, or TE buffer to elute the DNA from the filter paper. The liquid may be completely soaked up by the filter, this is OK. Cap the tube and leave at room temperature for 5 minutes. 3) Centrifuge the tube at high speed for 1 minute to collect the liquid, or use a pipette and "compress" the filter paper at the bottom of the tube to squeeze out as much liquid as possible. Collect the liquid in a new tube, and then use DH5a competent cells to amplify the plasmid, use the suitable antibiotic to select for positive clones.
Is the 5'LTR wildtype? For the 3' LTR, is the U3 region deleted?
This is not the wildtype 5'LTR. The U3 region is removed.
How does lentiviral delivery of siRNA differ from shRNA?
siRNA and shRNA have the same intention and they are to silent gene expression by cleaving the target mRNA. However, their differences are as follows: a) siRNA (small interfering RNA) is a linear sequence that binds to the target mRNA and cleaves it, preventing the unwanted protein from being made b) shRNA (short hairpin RNA) is a DNA construct encoding a sequence of single stranded RNA and its complement, separated by a stuffer fragment, allowing the RNA molecule to fold back on itself, creating a hairpin loop Traditionally, siRNA is made synthetically and introduced into the cells as is which is short lived; whereas shRNA is cloned into a vector and then introduced cell's genome, which can prolong expression. However, shRNA poses a difficulty on amplification and sequencing and thus it is not easy to construct. abm has come up with a novel technology where we combined the ability to clone siRNA into a vector, but with the same effect as shRNA. With the help of a dual convergent promoter vector system, the sense and antisense strands of the siRNA are expressed by two different promoters rather than in a hairpin loop, which thus avoids any possible recombination events that can occur. However, the siRNA sequence is expressed in both directions, and therefore mimicks the actions of shRNA.
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