CRISPR/Cas projects initiated with the core will have PCR amplicons generated by the core for Sanger sequencing. Mice can be genotyped by the investigator and the core can perform subsequent Sanger sequencing. Alternatively, the core can perform the initial genotyping and investigators can add on the Sanger Sequencing service to sequence confirm desired alleles before starting to breed founder animals or to verify alleles in N1 animals. The core will analyze the sequencing traces and repeat the sequencing if the results are ambiguous. Tail clips, ear punches, or purified DNA samples can be submitted.
What Will Happen
If the core initially performed genotyping on the samples that will be Sanger sequenced, no additional action is needed by the investigator. The additional services will be added on to the existing genotyping request.
- For stand-alone sequencing requests, 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 Sanger sequencing 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 Sanger sequencing synthesized by an approved vendor. Primers that were used for previous genotyping by the core will be used if appropriate for Sanger sequencing.
- The core will contact the investigator when it is ready to accept samples. Please do not submit 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 Sanger sequencing is completed, the investigator can arrange with the core to have aliquots of the DNA for their own sequencing, if desired.
- The core will perform standard PCR on the extracted DNA samples for the designed Sanger sequencing 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.
- 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 investigator 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 investigator 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.
- If any sample fails to produce a quality trace by Sanger sequencing, repeat Sanger sequencing attempts will be submitted until the core can accurately assess the sequence for the sample at the specific locus.
- 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.