HOW TO DO DILUTION SERIES FOR QPCR SERIES

Consider optimizing primer concentrations or T a (see Assay Optimization and Validation). Test the primers of probe assays in a SYBR В® Green I qPCR mix to verify amplification. Measure the working concentration of the oligo and visually inspect fluorescent molecules to confirm that these are labeled. Ensure that the oligo quality was correct by contacting the oligo vendor. Repeated cycles of freeze thaw can also affect oligo performance and therefore all oligos at a stock concentration (usually 100 ОјM) should be aliquotted and stored at -20 вЊ, or -80 вЊ for the long term.Дuring the troubleshooting phase it is critical to verify that the correct sequence was ordered by returning to the target sequence and confirming that the oligo sequences are indeed present. Resuspend oligos by heating the oligo to 90 вЊ for 5 min and then mixing well. Confirm the solution is the concentration expected.Ensure that all DNA is resuspended prior to use.On receipt of a lyophilized oligo it is critical to: An assay run under sub-optimal conditions for the oligo, or using a poor design may yield some data, however this may not reflect the genuine biology under consideration. Oligos may cause problems if they are an incorrect sequence or poorly designed, run at a sub-optimal concentration, sub-optimal Ta, or are inadequately labeled or quenched (for probes). This is particularly relevant to some of the 2Г— buffers such as KiCqStart В® that are more viscous than normal PCR buffers. Ensure that all components are carefully thawed and mixed well and that the experiment master mix is very well mixed before aliquoting to samples. It is good laboratory practice to ensure that sufficient reaction master mix is prepared for all samples that are to be run together. It is also essential to review the instructions provided with each master mix because they specify the recommended conditions that are optimized for the given enzyme, Hot Start mechanism and buffer components. Therefore, verify all assays in selected master mixes and on all desired instruments before making radical changes. Changing any one of these may result in different performance. When switching master mix products, it is critical to recognize that some assays are particularly sensitive to buffer composition/annealing temperature (T a)/primer concentration combinations.

HOW TO DO DILUTION SERIES FOR QPCR SERIES

If a new batch of reagent is being used, it is a useful precaution to run the new against the old before launching into a major series of experiments. Before repeating the experiment, check all components and their concentrations. Mistakes or problems with the master mix blend of reaction components may be the source of a catastrophic failure of amplification in all samples and positive controls. The cautionary tale of the postdoctoral fellow who ran several failing PCRs before realizing that the dNTPs were missing from the PCR master mix is a reminder that even the best over-worked scientists are vulnerable to simple errors. Checking the protocol (refer to Appendix A, Protocols, of this guide) and asking an experienced molecular biologist to review the experimental plan is important. The first step in any troubleshooting procedure is to check the protocol and repeat the experiment. The sources of these errors often remain unidentified. There are many possibilities for operator error. Developing a PCR or RT-PCR Troubleshooting Protocol Potential Sources of Error and/or Problems Operator Error

How to do dilution series for qpcr