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. For N1 animal genotyping, previously used genotyping designs for screening founders will be used to identify heterozygous animals inheriting the desired targeted allele. Tail clips, ear punches, or purified DNA samples can be submitted.
What Will Happen
- The investigator will initiate a project in iLab. An account number must be provided at this time.
- The investigator will specify the number of animals they have for genotyping and fill out the sample submission table. Samples should be labeled using the following convention: Use the 4-digit iLab request number (i.e. GERM-DL-2578) followed by a dash and a three digit number from 001 to the number of N samples submitted. For example, 2578-001, 2578-002, etc. Samples submitted without this naming convention will have these labels assigned to the samples accordingly.
- The core will have oligonucleotides for PCR synthesized by an approved vendor.
- The core will contact the investigator when it is ready to accept the samples. Please do not submit the samples until contacted by the core. Samples must be submitted in 1.5ml microfuge tubes to ABBR R851A. Each tube must be clearly labelled. The core will provide a cardboard tube box and will label the box with the correct project number.
- The core will extract DNA from the supplied tissues if needed and resuspend the DNA in nuclease-free water. After genotyping is completed, the investigator 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.
- Within two weeks of dropping off the specified number of samples, the core will provide the investigator with a genotyping report, including the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If desired, the Investigator can arrange a consultation meeting to discuss the genotyping results. The core will provide advice as to which founders to breed from founder genotyping.
- The investigator can decide whether they would like to continue with allele QA and have the core perform Sanger sequencing at an additional cost.
What to Expect
- The core will provide genotyping results from the previously designed schemes. If the PCR reactions fail to produce products with appropriate controls, new primers will be designed by the core and PCR will be repeated until genotyping results can be determined.
- Indel and point mutations are often too small to detect by conventional PCR. Sanger sequencing and trace analysis will need to be performed to identify and characterize alleles produced. The core will recommend samples for Sanger sequencing, and will send out prepared samples with approval from the investigator. Sanger sequencing reactions will occur an additional charge per sample.
- To verify any allele, Sanger sequencing of PCR products is necessary to verify the sequence at the target site(s). For N1 animals genotyped by the core, additional recommendations for samples for Sanger sequencing will be made. With approval from the investigator, new PCR amplicons will be generated and the core will send out prepared samples for Sanger sequencing. Sanger sequencing reactions will occur an additional charge per sample.
- Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at the founder generation. The core can make judgements as to which founders have targeted alleles, but the core cannot be completely certain without sequence confirmation.