Preparation of 96-Well Plate for Monochrome Multiplex Quantitative PCR (MMqPCR) for Telomere Length Measurement

To begin, place two 96-well plates, a loading trough, and multichannel pipette tips inside the PCR hood. After labeling each plate, turn plate 1 180 degrees so that the column numbers are upside down while leaving plate 2 facing the technician. Then pour the five milliliter tube of the prepared MM qPCR master mix into the loading trough.

Use a pipet tip to ensure all the solution from the tube is transferred. Attach tips to the multichannel pipette, pressing at the base of each tip to secure a tight seal. Using reverse pipetting, dispense the master mix into either all even or all odd columns first, alternating between the two plates.

After vortexing and centrifuging the PCR strips, arrange them in the tube rack in the order they will be placed on the plates. Now turn both plates 180 degrees so that the column numbers are reversed in relation to the technician. Use the multichannel pipette to resuspend solutions in the PCR strips.

Using reverse pipetting, fill the first three columns of each plate with PCR strip A, alternating between even or odd columns like the master mix placement. Using a 200 microliter pipette set to 90 microliters, thoroughly resuspend the seventh standard dilution tube in PCR strip S and fill the fourth through sixth columns on the plates. After filling the plates, gently tap them on the bench top to ensure the liquid in the wells settles at the bottom.

Then cover the tops of the plates with sealing films, pressing down on all edges of the film with fingertips to ensure a tight seal. To mix the contents of the plates, swirl them on the hood top for 30 seconds. Then place the sealed plates in the plate spinner for two minutes with the well openings facing the center.

Now place the plates in the thermocycler ensuring the column numbers are legible. Clean the top of each plate with a clean tissue before closing the thermocycler top. Then click the Start Run button on the thermocycler software for both machines.

When prompted, enter a title for the analysis files, which will be used to label and track the data from this experiment.