Bacterial Agar Stab
Bacterial stab of the custom plasmid.
|Unit quantity||1 Clone|
|I want to learn more about your ready to inject BAC purification services. I have 2 BAC clones that need to be isolated, digested (linearized), and purified for pronuclear injection in mice.|
Though the BAC is much larger than regular plasmid, we can still offer this service as long as the BAC is under 15kb. For BAC service, a special quotation has to be offered, depending on the nature of a particular plasmid. The digestion will cost another $100~200/100ug depending which enzymes to be used as some enzymes are more expensive than others.
|How much plasmid do I need to send you? How do you want it to be sent?|
Please send in at least 1 μg of plasmid DNA or your transformed bacteria. Information for the host bacteria is required if you are sending transformed bacteria with your plasmids. Also, please provide the antibiotic selection marker if available.
|What sort of information do I need to provide for the service?|
1. Number of plasmid. 2. Amount of DNA for each plasmid. 3. Plasmid map to check for transformation strain compatibility. 4. Antibiotic for proper plasmid selection. 5. Insert name so it can be confirmed to not be toxic or pathogenic. 6. High or low copy plasmid.
|For the Antibiotic Switching Service, can I change the gene to a resistance marker other than Puromycin or Neomycin?|
If you would like other resistance markers, we would need the customer to provide the plasmid containing the marker, as well as the full sequence, and we can then look into details of the marker and see if it would be possible. Please email [email protected] for further information.
|Can I send my plasmid as bacterial stocks?|
Typically, we request to receive the plasmids in liquid format as purified DNA, since this is most stable. However, bacterial stocks would also be suitable, provided that they are shipped to us within 2-3 days of preparation to ensure that the bacteria retain the plasmid.
|What is the lead time for a knockout service?|
4-5 months for a single gene knock-in.
|What should customers provide for this service?|
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.
|Can this service be performed for canine derived cells?|
Yes, we can use sgRNA targeting canine genome to make stable KI cell lines. Please provide the specific cell medium for canine cell culture.
|What information should I provide to get a quote?|
Please complete the Service Questionnaire form under the documents section above and email to [email protected]
|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.
|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.
|What are the deliverables?|
- Genetically Engineered Cell Line (up to 2 vials 1x10^6 cells/vial) - Custom Report
|Can you offer this service in bacterial cells?|
We offer CRISPR knockout and knockin on a custom inquiry basis. Please contact [email protected] with your project details.
|How is KI confirmed?|
We perform PCR and Sanger sequencing of the locus of interest to confirm the knockin. For even greater certainty, we also offer Next Generation Sequencing confirmation (IA00100) to confirm the absence of WT alleles. Being much more sensitive, NGS screening is ideal for detecting complete editing of multiple copy genes, or polyploid cell lines.
- DiVenere, A. M.. "Effective Antisense Design Using An Ensemble of Energetically Sub-Optimal Secondary mRNA Structures" University of Connecticut - Storrs UNIVERSITY SCHOLAR PROJECTS : (0). Application: Plasmid Amplification.
- Si, Y., Yang, Z., Ge, Q. "doi:10" 1186/s11658-019-0175-8 : (2019). DOI: 10.1186/s11658-019-0175-8.