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1. BLAST: BLAST® (Basic Local Alignment Search Tool) is a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA.

2. Entrez Gene: A Directory of Genes.

3. The Quantitative PCR Primer Database : (QPPD) provides information about primers and probes that can be used to quantitate human and mouse mRNA by reverse transcription polymerase chain reaction (RT–PCR) assays.

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Primer Design

 
 

Successful primer design depends on a number of factors. Presented here are some guidelines for designing PCR primers.

 

1. A primer length of 18-22 bases is effective in most cases.
2. Do not design primers with 3’ self complementarity; they will anneal to each other forming problematic primer-dimers.
3. The melting temperatures (Tm) of primers should be between 65 and 70 deg C. This temperature should be calculated for the region of specific hybridization.
4. The PCR annealing temperature should generally be 10 – 15 degrees lower than the Tm.
5. Avoid extended segments of a single nucleotide – aim for a balance of nucleotide types.
6. Generally, if the 3’ end is AT rich, it will be less likely to misprime.
7. Avoid internal hairpins – particularly at the 3’ end.

 

   

 

 

 

 

 

    
 

 

 

 
 
 
 
   
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