PRIMER_MISPRIMING_LIBRARY=/path/to/human_repeat_masked.lib PRIMER_MAX_MISPRIMING=12.00 # Maximum allowed mispriming score PRIMER_MAX_END_MISPRIMING=6.00 # Max mispriming score in last 5 bases : The mispriming scoring is more stringent. For highly repetitive targets, increase PRIMER_MAX_MISPRIMING to 15.0 . 6. Product Size Control PRIMER_PRODUCT_SIZE_RANGE=100-300 PRIMER_PRODUCT_OPT_SIZE=200 7. Internal Oligo (Probe) Parameters If PRIMER_TASK=pick_detection_primers , you can specify probe constraints.
PRIMER_MAX_MISPRIMING=12.0 PRIMER_MAX_END_MISPRIMING=6.0 PRIMER_NUM_RETURN=5 Running Primer3 v0.4.0 Save your input as input.txt . Then run: primer3 input -version 0.4.0-
PRIMER_OPT_SIZE=20 PRIMER_MIN_SIZE=18 PRIMER_MAX_SIZE=27 PRIMER_OPT_TM=60.0 PRIMER_MIN_TM=57.0 PRIMER_MAX_TM=63.0 PRIMER_MAX_DIFF_TM=3.0 Avoid 3' instability and low-complexity regions. PRIMER_MISPRIMING_LIBRARY=/path/to/human_repeat_masked