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Frequently Asked Questions: Stable Knockout Cell Line Generation

1. Will CRISPR keep cutting the chromosome after the gene is edited?
2. Do you provide knock-in cell line generation service?
3. 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)?
4. What are the deliverables?
5. How many sgRNAs are designed and synthesized for this service? Are pooled sgRNAs used?


1. Will CRISPR keep cutting the chromosome after the gene is edited?

CRISPR is sensitive to mismatches, so it is unlikely the CRISPR will keep cutting the chromosome after the gene is edited.

2. Do you provide knock-in cell line generation service?

Yes, but this will be at an additional charge and will be subject to a custom quotation.

3. 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)?

Yes, that is one method used. Other methods include Sanger sequencing, and western blotting if the antibody is provided by the customer.

4. What are the deliverables?

- 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.

5. How many sgRNAs are designed and synthesized for this service? Are pooled sgRNAs used?

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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