Use a computer program such as Primer Express® (Applied Biosystems) or equivalent to design primers. Alternatively already designed primers in several open access databases may be used (see Chapter 5). For analysis of cDNA from messenger RNA, design PCR primers to span one intron. Should any genomic DNA be present following the DNase treatment, the thermal denaturation protocol will detect two different PCR products, the shorter cDNA and the much longer genomic DNA. A convenient method to determine where the intron/exon junctions exist in a cDNA sequence is to BLAST the cDNA sequence of your gene of interest against the genomic sequence for the same gene. The BLAST report will locate the sequence of the intron/exon junctions. Adhere to the rules of primer design listed in Table 7.1. Dissolve primers in molecular biology grade water and store at -20°C.
Table 7.1 Design criteria for real-time PCR primers, SYBR® Green detection
Primer length 18-24 nt
Amplicon length <250 bp (ideally <150 bp)
Both sense and anti-sense primers should have a Tm <2°C of each other Primer Tm 50-60°C (ideal range 55-59°C) No consecutive runs of the same nucleotide more than six times No runs of more than three consecutive Gs
% GC content of primers ~50% (no less than 35% and no more than 65%) No 3' GC clamp on primers (i.e. GG, CC, CG or GC) <2 GC in the last five nucleotides of the 3' end of the primer
See also Chapter 5 for more considerations on primer design.
An example of a PCR set-up sheet. The numbers (1 to 6) represent different samples of cDNA. The numbers on the top of the columns (e.g. 313/232) represent the pair of primers.
Working stock solutions of primers are diluted to 50-100 pM and stored at -20°C.
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