Genotyping Sample Clauses

Genotyping. MAYO will use reasonable efforts to provide LICENSEE, subject to availability, breeding records and a protocol useful in genotyping the offspring of the Stock. Any breeding records or nucleic acid probes provided to LICENSEE by MAYO under this LSRA shall be used solely for genotyping the Licensed Technology.

Genotyping. All samples were digested as described by Xxxxxxxxx et al (Xxxxxxxxx et al. 1994). Single strand conformation polymorphism analysis (SSCP) of products was used for genotyping and mutation detection of all except the Scandinavian cases and relatives. A 50:50 mix of sample and 95% deionised formamide, 10mM NaOH loading buffer was denatured at 95°C for 3 minutes and snap cooled on ice. Electrophoresis was in 0.5xMDE gel, 0.6xTBE buffer at 8W and 25°C for 6 hours. Products were visualised after SSCP by silver staining. Of the 955 samples screened, 4 (all controls from Scandinavia) persistently had non-specific banding, making the gel difficult to interpret. Direct sequencing and attempts at subcloning of these samples were unsuccessful. Southern hybridisation by standard methods was used to determine the specific banding of these samples. Probe was prepared by PCR amplification of a previously sequenced sample from a control individual, known to be homozygous for the short NFH allele, and labelled by the random priming method. Filters were hybridised at high stringency. Two of these samples had band shifts consistent with deletions. Because of the difficulty in sequencing or subcloning these samples, they were electrophoresed in 10% acrylamide, visualised by ethidium bromide staining and directly sequenced from the gel. The Scandinavian cases and relatives were screened by PCR-digestion as above, followed by restriction fragment length polymorphism analysis (RFLP) by electrophoresis in 2% Metaphor agarose (FMC), 1xTBE buffer for 7 hours at 130V. Visualisation was by ethidium bromide staining.

Genotyping. Ear samples were taken from ephrin B2-/- mice at weaning and genotyping performed to establish their cre and floxed ephrin B2 status. In addition, ear samples were taken from wild-type and ephrin B2-/- mice after tamoxifen treatment to confirm successful deletion of ephrin B2. DNA was extracted using the ‘hotshot’ method described by Xxxxxx et al. (209). Cell lysis buffer (75µl) was added to tubes containing ear sample, and heated to 95ºC for 30 minutes with vortexing for 1 minute half way through incubation. Neutralization buffer (75µl) was then added to the tubes and they were stored at -20ºC. For PCR reactions, 2µl DNA was added into PCR tubes with: 12.5µl Taq PCR master- mix (Qiagen, Inc., Valencia, CA, US), 2.5µl Coral load 10x (Qiagen, Inc.), 9.5µl DNAse free water (Qiagen, Inc.), and 0.5µl primer. The PCR conditions consisted of: an initial hold-step at 95ºC for 5 mins, followed by 35 cycles of; 95ºC for 30 seconds, 63ºC (50ºC for knockout genotyping) for 30 seconds and 72ºC for 30 seconds. PCR products were run on a 3% agarose gel.

Genotyping. Blood was drawn into EDTA-containing tubes, and DNA was extracted using the Invitrogen pure clean genomic DNA blood extraction kit. Genotyping of DBH rs1611115 was performed using a validated Taqman SNP assay (C-2535789_10) from Applied