Addgene: Protocol - How To Design Primers
Addgene: Protocol - How To Design Primers
Addgene: Protocol - How To Design Primers
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The size of the primer is very important as well. Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other han
primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too long (> 30-mer primers) or too short. Short pri
produce inaccurate, nonspeci]c DNA ampli]cation product, and long primers result in a slower hybridizing rate. On average, the DNA fragment t
needs to be ampli]ed should be within 1-10 kB in size.
The structure of the primer should be relatively simple and contain no internal secondary structure to avoid internal folding. One also needs to a
primer-primer annealing which creates primer dimers and disrupts the ampli]cation process. When designing, if unsure about what nucleotide t
a certain position within the primer, one can include more than one nucleotide at that position termed a mixed site. One can also use a nucleotid
molecular insert (inosine) instead of a regular nucleotide for broader pairing capabilities.
Taking into consideration the information above, primers should generally have the following properties:
Note: If you will be including a restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp" should be added upstream
for the enzyme to cleave eDciently (e.g. GCGGCG-restriction site-your sequence).