QRT-PCR protocol General notes: Consistency is crucial to the accuracy of QRT-PCR, so the extra steps in this protocol are necessary. In general, be extra careful to treat every sample the same. Always use filter tips, and do no aspirate when pipetting (only go down to the first stop). Avoid bubble formation, which can accelerate the degradation of the cDNA. The SYBR Green is photosensitive, so the mixing is done in the “dark” room in Judy Manning’s lab, which is also the location of the iCycler machine. For the final spin of the 96-well plate, wrap the plate in foil before bringing it into the lab, and minimize exposure to light. Samples are done in triplicate to ensure that the technique is good. Before beginning protocol 1. Determine what templates will be used, and which will be used in the dilution series. At least one template should have a dilution series of five 10-fold dilutions. 2. For the primer set being used, determine the best annealing temperature by doing end-point PCR using a gradient for the annealing temperature. Pick the highest temperature with the best band. Use this temperature for the QRT-PCR reaction. Because of this, different primer sets must be done in separate iCycler runs, unless the best annealing temperature is the same for two sets. 3. Sign up for the iCycler ahead of time. The sign-up sheet is currently at the end of the McFall-Ngai lab bench on the left side of the aisle, as you face the windows. 4. It is a good idea to get the iCycler program and well designations done before starting work with the samples. Protocol: 1. Make master mix: Multiply the volumes per reaction by the number of reactions, and add 10%. Component 2x iQ SYBR green Forward Primer (10 uM) Reverse Primer (10 uM) Sterile Distilled H2O Volume added per reaction 12.5 ul 0.5 ul 0.5 ul 10.5 ul Total volume 24.0 ul Note: the amount of primers used will vary with the stock concentration. This assumes that 1 ul template is added per reaction. If this is not the case, adjust the volumes accordingly. 2. Vortex mix briefly, then do a short spin to remove any bubbles. 3. Prepare PCR tubes with 3 ul of each template. 5. Add 72 ul of master mix to each tube. 6. Vortex and spin tubes briefly. 7. Place 23 ul of each sample in the appropriate wells in a 96-well plate (less than the full amount is used to make sure that there is enough for all three wells, and to avoid bubbles). Make sure to put the samples in the correct well, as only those wells designated by the program will be read and recorded. 8. Carefully put clear cover on the plate. Use paddle to smooth down – do not touch cover. 9. Wrap plate in foil. Find similarly filled plate (there’s probably a pile) for a balance. Take into main lab, and spin down in Sorval Legend RT at *** for **** minutes. 10. Return to iCycler room. Keep plate in cold block. Begin iCycler run, and when the block gets up to temperature, pause the program and place the plate in the block. Well A1 goes in the top left as you are facing the machine. Unpause the program, and allow to run. Do not turn on the lights in the room until the run is over.