CRISPR Stable Knockout Cell Line Generation
Cat. No.
C208
Unit
2 Vials
Price
Inquiry
| Cat. No. | C208 |
| Name | CRISPR Stable Knockout Cell Line Generation |
| 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. C208 |
Print/Download Datasheet
| What is the lead time for a knockout service? | |
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3-4 months for a single gene knockout.
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| What should customers provide for this service? | |
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Please supply the cells for this service. For frozen cells, please ship at least 2 vials (at least 10^6 each) on dry ice. An alternative method is to send us two T25 flask of live cells per sample, at 90% confluency. The flasks should be filled with complete medium without any air bubbles and at room temperature. Please ensure that the sterilization procedure is strictly observed. To avoid delays over the weekend, we recommend shipping the cells on a Monday or a Tuesday. Frozen cells are preferred over live cells.
If your cell line already contains an antibiotic resistance marker, please specify. Our vectors have puromycin resistance by default.
If your cells require medium other than DMEM and RPMI, we will also require 1L of the specified medium and any applicable growth factors or supplements to be provided by you for the project. Please submit all components of the complete media (e.g. if growth factors, cytokines etc. are applicable) individually to eliminate potential degradation of components in the media during transit. Kindly include instructions for making the complete media in your shipment. Additionally, please provide at least 5 x coated 6-well plates and 10 x T25 flasks if the cells require specially treated culture vessels. Once you have shipped your cells, please forward us the tracking number for custom clearance. Information on how to ship cell samples to abm can be found on our support page: https://www.abmgood.com/Technical-Support.html
Please place an order first prior to submitting your samples. All samples received must have the order confirmation number indicated. Any samples received without this piece of information will be disposed off immediately upon receipt to ensure that all customer information is held in strict confidence.
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| Can this service be performed for canine derived cells? | |
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Yes, we can use sgRNA targeting canine genome to make stable KO cell lines. Please provide the specific cell medium for canine cell culture.
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How should I handle live cells once I receive them? |
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Please refer to our Cell Handling and Thawing Guidelines for detailed instructions on receiving, thawing, and culturing live cells: |
| What information should I provide to get a quote? | |
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Please complete the Service Questionnaire form under the documents section above and email to quotes@abmgood.com.
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| Will CRISPR keep cutting the chromosome after the gene is edited? | |
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CRISPR is sensitive to mismatches, so it is unlikely the CRISPR will keep cutting the chromosome after the gene is edited.
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| Is Nuclease or Nickase used? Can you use the double nickase approach? | |
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The approach used for the above service is sgRNA with Cas9 Nuclease, relying on NHEJ repair without a repair template. The Nickase approach is available but this will be at an additional charge and will be subject to a custom quotation as a different strategy will need to be devised.
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| Do you provide knock-in cell line generation service? | |
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Yes, but this will be at an additional charge and will be subject to a custom quotation.
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| How can you detect a mutation if there is no selection (by PCR using primers flanking the modified site or the T7 endonuclease I assay)? | |
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Yes, that is one method used. Other methods include Sanger sequencing, and western blotting if the antibody is provided by the customer.
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| What are the deliverables? | |
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- Reports on the Cas9-sgRNA vectors used for KO and the sgRNA sequence.
- at least 1 clone, 2 vials per clone
- Sanger sequencing data showing that there are frameshift mutations in each clone
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| How many sgRNAs are designed and synthesized for this service? Are pooled sgRNAs used? | |
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We design 3 sgRNAs at the time of order placement, however only 1 sgRNA is used to achieve knockout. In the case that the first sgRNA does not allow for success, we would try another from the set of 3 synthesized sgRNAs.
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| Can you offer off-target site prediction service and off-target site analysis service (Sanger sequence)? | |
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When we design the sgRNAs, the design software will predict the number of off-targets, which is usually 0 for the sgRNAs that we select.
Off-target analysis: This will need to be done by NGS, whole genome sequencing or exome sequencing, which would require comparing the control (before) sample to the genome edited sample (after). If this is a desired option, we can provide a quote for it, but it will likely be at least $2200 for analyzing 1-2 clones by Exome Seq, and higher for whole genome sequencing.
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| Can you offer this service in bacterial cells? | |
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We offer CRISPR knockout and knockin on a custom inquiry basis. Please contact technical@abmgood.com with your project details.
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| How is KO confirmed? | |
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We perform Sanger sequencing of the locus of interest both to determine the nature of the INDEL in the edited genes to guarantee frameshift silencing, and to confirm that the genome is edited. For even greater certainty, we also offer Next Generation Sequencing confirmation (AI00100), which can detect WT alleles down to 10% of total. Being much more sensitive, NGS screening is ideal for detecting complete editing of multiple copy genes, or polyploid cell lines.
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What is your warranty or return policy? |
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Our warranty and return policy is outlined in abm’s Terms and Conditions, including details on product quality, limitations, and claims. For additional questions, our Order team is happy to assist and can be reached at order@abmgood.com. |
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How many times can cells divide? |
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The number of times cells can divide depends on the cell type:
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Do I need Applied Cell Extracellular Matrix (G422) if I am using PriCoat™ flasks? |
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Please refer to the Growth Conditions section of your specific product page. This section will indicate whether Applied Cell Extracellular Matrix (G422), PriCoat™ flasks, or both are recommended for optimal cell attachment and growth, as requirements may vary by cell type. |
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