Realtime PCR

Perform triplicate PCRs per gene, per cDNA sample. Most real-time PCR instruments are configured with a 96-well block. Use a 'PCR set-up sheet' (Figure 7.1) to organize the pipetting of the plate as well as to coordinate the data during the analysis.

29. Prepare the following master mix, accounting for 1-2 additional reactions per gene:

Ingredient Per reaction

2X SYBR® Green reagent 12.5 pl

Forward/Reverse primer mix 0.125 pl (50 pM each)

Molecular biology grade water 7.375 pl

For x reactions x x 12.5 pl x x 0.125 pl x x 7.375 pl

30. Dilute the cDNA 1:50 or 1:100 by first placing molecular biology grade water into a disposable sterile basin. Add 99 ^l of the water into the PCR strip tube using a multichannel pipette.

31. Add 1 ^l of cDNA to the labeled strip tubes using a multichannel pipette (e.g. Rainin L8-10). Use a multichannel pipette (e.g. Rainin L8-200) to mix the solution 20 times. For efficient mixing, set the pipette at 75% of the solution's volume (75 ^l in this example).

Refer to section on critical steps

32. Recap the undiluted cDNA with new strip caps to prevent cross contamination.

33. Use a fine tip marker to mark the plate to the location of the different master mixes similar to the set-up sheet in Figure 7.1.

34. Use the repeating pipette (Rainin, E12-20) to add 20 ^l of master mix to each sample. Add one row at a time.

35. Add 5 ^l of dilute cDNA to each of the wells using a multichannel pipette.

36. Add the optical adhesive cover and seal using the sealing tool. Perform a brief spin (up to 1500 RPM) on a centrifuge equipped with a 96 well plate adapter.

37. Perform PCR using the real-time instrument per the manufacturers' protocol. Typically, 40 cycles of 15 sec at 95 °C and 60 sec at 60°C followed by the thermal dissociation protocol.

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