Sample and Master Mix Preparation for Telomere Length Measurement Using Monochrome Multiplex Quantitative PCR (MMqPCR)

Begin by adding genomic DNA samples extracted from peripheral blood mononuclear cells to PCR strips A, B, and C respectively. Then vortex the thawed controlled DNA aliquot for 30 seconds. After briefly centrifuging the tube, aspirate the full volume into the first tube in the standard curve PCR tube strip.

Next, add 70 microliters of PCR grade water into the second through eight tubes of the standard curve PCR strip. Then resuspend control DNA in the first tube and aspirate 70 microliters into the second tube. Wait for 30 seconds and resuspend the solution in the second tube.

Collect the master mix reagents and allow them to reach room temperature in the PCR hood. Then add one microliter of the cyber green aliquot to the buffer aliquot. Vortex all aliquots for 10 seconds and centrifuge them for five seconds.

Then add the specified amounts of all master mix reagents and primers into the five milliliter betaine tube. After taking out the DNA polymerase from minus 20 degrees Celsius, vortex it for 10 seconds, and then centrifuge for five seconds. Once centrifuged, slowly add 128 microliters to the master mix.

Vortex the five milliliter master mix tube for 30 seconds and then set it aside.