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Table 2 Primer Design Parameters Used in the Four Passes of Primer3.

From: Optimus Primer: A PCR enrichment primer design program for next-generation sequencing of human exonic regions

  First Pass Second Pass Third Pass Fourth Pass
Optimum Size (BP) 25 25 25 25
Minimum Size (BP) 20 20 18 20
Maximum Size (BP) 30 30 33 30
OptimumTM (°C) 60 60 60 60
Minimum TM (°C) 55 55 50 55
Maximum TM (°C) 65 65 70 65
Optimum GC% 50 50 50 50
Minimum GC% 40 40 35 40
Maximum GC% 60 60 65 60
GC CLAMP Yes No No Yes
NUM RETURN 1 1 1 1
PRODUCT SIZE RANGE (BP) 400-600 400-600 400-600 400-600
Repeat Masking Yes Yes Yes No
SNP Masking Yes Yes Yes Yes
  1. Optimus Primer allows the user to specify up to four sets of PCR primer design parameter simultaneously. Amplicons for which no primers are designed in the first pass are automatically passed on to the next pass. Above are the four sets parameters used to design primers in our validation experiments.