Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

 

Primer and Probe Design for Quantitative Assays:


Primer and probe design software from ABI (ABI PRISM Primer Express) is available at both computer stations in the DNA Sequencing and the Core Genomics Facilities. When designing only PCR primer pairs, other general primer design programs could be used such as: Primer Quest available free of charge at: http://biotools.idtdna.com/primerquest/ and Primer Select by DNAstar (available at Core Genomics Facility computer station).


Please refer to the following ABI recommended guidelines when designing primers / probes for Quantitative assays:
· select the probe first and design the primers as close as possible to the probe without overlapping it. Amplicons of 50 to 150 bp are strongly recommended. If absolutely necessary product size could be increased up to 200-250bp at most
· keep primer/probe GC content within 30-80%
· avoid runs of identical nucleotides, this is especially true for guanine, where runs of four or more Gs should be avoided

When designing primers:
· Tm should be within 58°C to 60°C
· the last 5 bases at the 3 prime end should have no more than two G's or C's
· keep the annealing Ts of the primers as close as possible
· select primer pairs with minimal number of potential primer dimers and primer hairpins as possible

When designing probes:
· Tm should be within 68°C to 70°C
· no Gs on the 5’ end
· select the strand that gives the probe more C than G bases
· make the TaqMan MGB probes as short as possible, without being shorter than 13 nucleotides
Before to proceeding with real time PCR, it is necessary to test the primers on a PCR reaction to ensure that the primers amplify the gene of interest at the right size and that the primers are specific (i.e. no other bands present on the gel except that of the expected size). In doing this, it is important to run your test PCR under conditions as close as possible to those for real time PCR (refer to the table below). Also, the best results are achieved with magnesium concentration at 3mM and with 0.025 U/ul of Taq.

Table 4: Typical Real Time PCR stage Parameters


 

Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

 RRC Core Genomics Facility
University of Illinois at Chicago
2003