• DNA:                                        As per requirement
• True allele PCR Premix :     4.5 µL
• Sterile ddH₂O:                        - µL
• Total Volume :                       7.0 µL

• Dispense the aliquot to each PCR well and then add 0.5 µL of respective primers
• Give a short spin
• Cover the plate with 8 tube strips
• Place in the PCR with the fallowing reaction program
  • 95° C for 12 min
  • 10 cycles of
    • 94° C for 15 sec
    • 55° C for 15 sec
    • 72° C for 30 sec
  • 20 cycles of
    • 89° C for 15 sec
    • 55° C for 15 sec
    • 72° C for 30 sec
  • Final extension of 72° C for 10 min
  • Hold at 4° C forever
• For multiplexing we can pool the PCR products in the ratio of 1:1:2 (FAM:VIC:NED)
• Make the master mix of
  • HiDi-Formamide:           9 µL
  • GS 500 LIZ:                   0.5 µL
  • Total:                             9.5 µL
• Add the 9.5 µL HiDi-LIZ mix to each PCR well and add 1.0 µL (or 2 µL) of pooled PCR product
• Short spin, denature (95° C for 5 min) and snap chill (4° C for 5 min)
• Make plate record for fragment analysis and start the run (with G5 Dye set in 3500)
• Analyze the data using Genemapper software