genotypinG

experimental objectives

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented Genotyping protocol. 

methodology

Primers were designed and obtained using the Oligosynthesis Quick Instruction. Primers annealed to complementary sequences in the murine genome thereby assessing the presence of transgenic insertion. The presence of inserted transgene yielded amplicons of 595 bp (generated by amplification of DNA with Primers A and C), internal control murine sequence generated amplicons of 324 bp (Primers B and D). PCR was performed using 2 µl of template DNA, 0.3 µl Primer A, 0.3 µl Primer B, 0.3 µl Primer C, 0.3 µl Primer D (all 20 µM). Total reaction volume was 15 µl made up using 7.5 µl of 2x MyTaq HS Mix (Bioline) and 4.3 µl of distilled water. Touchdown thermocycling conditions were 94°C, 2 minute; [94°C, 20 seconds; 65°C, 15 seconds; 68°C, 10 seconds] x10 with annealing temperature decreasing 1 degree every 2 cycles from 65°C to 61°C, followed by [94°C, 15 seconds; 60°C, 15 seconds; 72°C, 10 seconds] x28 with a final 2 minute incubation at 72°C. Successful amplification was confirmed by gel electrophoresis. 

results

Amplification of mouse-derived DNA was successfully achieved in a four primer assay. Internal controls and/or transgeneic alleles were identified in all samples (Figure 1) allowing unambiguous genetic profiling. 

Figure 1 Ten mouse-derived DNA samples were assayed for presence of transgene alleles. Four samples contained the transgene with the remainder being wildtype. 

Figure 1 Ten mouse-derived DNA samples were assayed for presence of transgene alleles. Four samples contained the transgene with the remainder being wildtype. 


VALIDATION DATE:  October 2015