qPCR 384-well plate loading training

Now that the RT is achieved, the next step will be to perform the PCR itself.

A few important comments :

  • Loading a 384-well plate is hard, so that we will perform training before loading the plate that will be used for the qPCR. Do not distract those who are loading the samples into the plate, as it will increase the risk of contamination in the samples being loaded as well as those of other groups.
  • In the case of qPCR, each reaction (each sample with each primer pair) is performed in triplicates in order to make sure that the amplification is accurate. Indeed, one of the quality control steps we make when analyzing qPCR data consists of making sure that the variation between technical replicates is minimal.
  • To gain time when loading the full plate, we will ask you to produce mixes containing all the Master mixes (DNA polymerase + buffer + dNTPs) + primers + cDNA. These mixes will then be spread into 3 wells (= technical replicate). This differs from the method usually used in the lab, in which the loading time is longer for a small number of samples, consists of :
    1. Creating one mix for each primer pair without the cDNA (= Master mix + primers + water) ;
    2. Adding this mix to the required wells ;
    3. Adding each cDNA to the different samples.
  • Controls. As always in molecular biology, controls are important. For each primer pair, you will thus perform a blank mix in which everything is added, except your sample, which is replaced by water. If an amplification reaction occurs in this sample, this suggests that the reagents are contaminated with gDNA and/or cDNA, or that the primers interact during the PCR reactions, leading to the creation of primer dimers.
  • The full protocol of the qPCR Master Mix used during this experiment (Takyon Master Mix, Eurogentec) is available through the following link : https://tinyurl.com/yv62eecz

Before performing the full experiment, let’s practice!

Although we will proceed slightly differently for the loading of your samples to the final qPCR plates, the usual way to load such a plate is to add the buffer mix (typically 8 µl) to the different wells in which to perform a reaction and, then, to add the cDNA. Here, we provided you with 11 used 384 well plates, 10 µl tips, water and buffer 1X (tubed labeled TB for “Training buffer”) in order for you to train to load the plate before the proper loading.

For each student :

  • Use the P10 pipette to add 8 µl of the buffer to all the wells of a row (1 to 24). Keep the same tip for all the row.
  • After the row is loaded, change tips.
  • Add 2 µl of water (= cDNA) to each well (for training, do not change your tip, but you’ll need to change it when loading the 384-well plate used for the RT-qPCR).