Common use of Genotyping Clause in Contracts

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 ▇▇▇▇▇▇ 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.