Real -Time PCR Tips & Tricks From The Life Lab
by Jennifer Hornstein - 05/24/12
- Reduce contamination risk with a separate area in your lab just for real-time PCR experiments—create an “RNA Zone” for sample prep extraction, external sample preparation, reaction mixture set-up, and running the real-time PCR instrument.
- Use Ambion RNaseZap® RNase Decontamination Solution to get rid of RNases and other contaminants prior to same prep procedures.
- Perform a bioinformatic evaluation of your target sequence before submitting the sequence into our online custom TaqMan® Assay design tool.
- Before resuspension of your lyophilized TaqMan® Assays, spin down the tube to ensure that you are resuspending everything in the tube.
- Make aliquots of your resuspended TaqMan® Assays to avoid repeated freeze/thaw cycles.
- Prevent cross contamination with filter tips or positive displacement pipettes.
- Cap the wells for No Template Control (NTC) prior to adding any sample cDNA/DNA or external standards during the total reaction preparation.
- Add DNA or RNA to your wells last to minimize cross contamination.
- Keep the bottom of your plate clean—use a support base for 96-well plates or a lint-free wipe/cloth for 384-well plates.
- Centrifuge your PCR plate before loading into the instrument to remove bubbles and ensure all reaction components are at the bottom of the well.
- When setting up and running replicate wells, make sure that your sample names, detector names, and detector tasks are the same.