To guarantee PCR efficiency, small PCR products are recommended. Most PrimerBank primers lead to amplicons in the size range of 100 - 250 bp. PCR efficiency is close to 100% in this range (supported by real-time PCR experiments). However, PCR efficiency may be reduced for much larger PCR products. . Less than 1% of PrimerBank primers lead to >400 bp amplicons. Please be cautious about PCR efficiency when these primers have to be used.

Sometimes it is desirable to select a primer pair that spans intron. In this way, genomic DNA contamination can be closely monitored. Usually a PrimerBank primer pair spans intron because a typical exon is quite small. The primer intron-spanning information will be included in the next version update.  If it is important for you to be sure that a primer pair spans intron, you may simply do a BLAST search of the primer pair sequences, or better the amplicon sequences (listed in the primer detail page) against the genomic sequences.

Agarose gel electrophoresis or melting curve analysis may not always reliably reflect PCR specificity. From our experience, bimodal melting curves are occasionally observed for long amplicons even when the PCRs are specific. The observed heterogeneity in melting temperature was due to internal sequence inhomogeneity (e.g. independently melting blocks of high and low GC content) rather than amplicon contamination. On the other hand, for short amplicons very weak bands migrating ahead of the major specific bands are occasionally observed on agarose gel. These weak bands are super-structured or single-stranded version of the specific amplicons in equilibrium state. Although gel electrophoresis or melting curve analysis alone may not be 100% reliable, the combination of both can always reveal PCR specificity in our experience.

Although all PrimerBank primers are designed to be gene specific, we still need to be very careful about PCR conditions. 

Non-specific primer extension of only a few bases at low temperature by DNA polymerase can easily lead to non-specific PCR amplifications. Therefore, hot-start PCR is STRONGLY recommended. In fact, I only do hot-start PCR in my experiments. I routinely use AmpliTaq Gold polymerase (Applied Biosystems) for hot-start PCR.

The annealing temperature may affect PCR specificity. To avoid non-specific PCR products, a high annealing temperature (the smaller one of the two Tm values from the primer pair) and a short annealing time are recommended.

If you still see non-specific bands, it could mean the primer pair in use is the problem. Although great care has been given to design PrimerBank primers, the success rate is not 100%. Less than 1% of the primers may have design problems (see our paper for detailed discussion). In this case, please try a different primer pair for the same gene.

Poor quality of PCR templates, primers, or reagents may lead to PCR failures. First, please include appropriate controls to eliminate these possibilities.

Some genes are expressed only in certain tissues. Please first read literature to make sure your genes are included in the cDNA templates. In our experience, this is the most likely cause for negative PCR results. If you are sure the genes are expressed, then try lowering the annealing temperature (to Tm - 5 °C) and increasing the annealing time to ensure sufficient primer annealing.

If you still could not see any PCR band, it could mean the primer pair in use is the problem. Although great care has been given to design PrimerBank primers, the success rate is not 100%. Less than 1% of the primers may have design problems (see our paper for detailed discussion). In this case, please try a different primer pair for the same gene.

PrimerBank was created by Dr. Xiaowei Wang at the Department of Molecular Biology, Massachusetts General Hospital in Boston. Besides PrimerBank, Dr. Wang have also developed other bioinformatics programs.

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