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Tips for the successful RealTime PCR

RealTime PCR, also known as quantitative or qPCR, is an effective tool to make the in-vitro amplification of specific DNA fragments visible without using an agarose gel. As the term implies, the amplification process can be analyzed while PCR is still proceeding (real time).

In RealTime PCR different fluorescent dyes and approaches are used to trace the progress of amplification during the PCR reaction and to get a strong increase in fluorescence during amplification.

For a successful RealTime PCR, remember the following tips:

- Consider the requirements of your thermal cycler, e.g. as far as the ROX concentration in your mastermix!

- When using probes, the length of the target fragment should be between 90 to 250 bp.

- To protect the stability, keep primers in a buffer at neutral pH.
DNA can be damaged after resuspension in water with reduced pH (especially if it was treated by DEPC) over time. We recommend using a buffered solution with a neutral pH or 1 mM EDTA in the stock solution.

- Aliquot your primers to prevent damage caused by repeated freezing and thawing. Thus, the risk of contamination of the stock solution is reduced.

- First, create a standard curve for each new primer pair to test the efficiency.

- Mix your reagents well before use!
By storaging in a freezer or refrigerator it comes to partial settling of individual component reagents (dyes, nucleotides, enzymes). To avoid an uneven distribution of reagents in your samples, good mixing is required.

- Avoid the formation of air bubbles when mixing, as these can interfere with fluorescence detection!

- Dilute the template (less is often more)!
Sometimes a more accurate measurement results from the fact that less template is used. The sensitivity of RealTime PCR is sufficient for this purpose! Disturbing influences of inhibitors from the purification step in the sample (for example, guanidine salts or ethanol) can thus be avoided.

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