CRISPR/Cas projects initiated with the core will be genotyped for desired genome editing events in founders using previously designed schemes or schemes approved by the core. users can add on Sanger Sequencing to sequence confirm desired alleles before starting to breed founder animals. For N1 animals Sanger sequencing will be offered as an add-on service to PCR genotyping. Previously used genotyping designs for screening founders will be used to identify heterozygous animals inheriting the desired targeted allele. PCR products will be cleaned up and sent out for Sanger sequencing. The core will analyze the sequencing traces and repeat the sequencing if the results are ambiguous.
What Will Happen
- The user will initiate a project in iLabs for N1 PCR genotyping and Sanger sequencing. The User will specify the number of animals they have for genotyping. An account number must be provided at this time.
- The user will collect tissue from the mice to be genotyped and arrange with the core to drop off the tissue.
- The core will have oligonucleotides for PCR synthesized by an approved vendor.
- The core will extract DNA from the supplied tissues and resuspend the DNA in nuclease-free water. After genotyping is completed, the user can arrange with the core to have aliquots of the DNA for their own genotyping, if desired.
- The core will perform standard PCR on the extracted DNA samples for the designed genotyping reactions.
- The core will clean up the PCR reactions with the anticipated product size for the desired allele and send out samples for Sanger sequencing. If necessary, the Core will gel extract and purify PCR samples with multiple bands to sequence the desired allele.
- The core will analyze the Sanger sequencing results to confirm the sequence of the desired allele.
- Within two weeks of dropping off the specified number of samples, the core will provide the user with a report, including the total number of animals analyzed, total number of animals sequenced, and total number of animals with desired allele. If desired, the user can arrange a consultation meeting to discuss the sequencing results.
What to Expect
- The core will provide sequencing results from the previously designed genotyping schemes. If the PCR reactions fail to produce products with appropriate controls, new primers will be designed and PCR repeated before any sequencing is attempted.
- Bioinformatics software is available to deconvolute heterozygous trace files containing indel and short HDR alleles in addition to wild-type sequence. This software will be used in place of traditional TA cloning to separate multiple PCR products for indel, epitope tag, point mutation, and conditional alleles. The core will not provide TA cloning services.