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High-throughput Primer Design Using Primer3
Help Copyright Notice and Disclaimer of Primer3 Comments and Suggestions Primer 3 Source Code

Input Sequence(s) and related data for batch processing:

Upload sequence file with FASTA format:
OR paste source sequence(s) below (5'->3', string of ACGTNacgtn -- other letters treated as N -- numbers and blanks ignored).
FASTA format ok. Please N-out undesirable sequence (vector, ALUs, LINEs, etc.) or use a
Mispriming Library (repeat library):
Upload file with sequence ID and target positions:
Pick left primer or use left primer in the right field.
Pick hybridization probe (internal oligo) or use oligo in the right field.
Pick right primer or use right primer in the right field (5'->3' on opposite strand).

Sequence Id: A string to identify your output.
Targets: E.g. 50,2 requires primers to surround the 2 bases at positions 50 and 51. Or mark the source sequence with [ and ]: e.g. ...ATCT[CCCC]TCAT.. means that primers must flank the central CCCC.
Excluded Regions: E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<CCCC>TCAT.. forbids primers in the central CCCC.
Product Size Min: Opt: Max:
Number To Return: Max 3' Stability:
Max Mispriming: Pair Max Mispriming:

General Primer Picking Conditions

Primer Size Min: Opt: Max:
Primer Tm Min: Opt: Max: Max Tm Difference:
Product Tm Min: Opt: Max:
Primer GC% Min: Opt: Max:
Max Self Complementarity: Max 3' Self Complementarity:
Max #N's: Max Poly-X:
Inside Target Penalty: Outside Target Penalty: Set Inside Target Penalty to allow primers inside a target.
First Base Index: CG Clamp:
Salt Concentration: Annealing Oligo Concentration: (Not the concentration of oligos in the reaction mix but of those annealing to template.)
Liberal Base Show Debuging Info

Other Per-Sequence Inputs

Included Region: E.g. 20,400: only pick primers in the 400 base region starting at position 20. Or use { and } in the source sequence to mark the beginning and end of the included region: e.g. in ATC{TTC...TCT}AT the included region is TTC...TCT.
Start Codon Position:

Sequence Quality

Min Sequence Quality:
Min End Sequence Quality:
Sequence Quality Range Min:
Sequence Quality Range Max:

Objective Function Penalty Weights for Primers

Tm Lt: Gt:
Size Lt: Gt:
GC% Lt: Gt:
Self Complementarity
3' Self Complementarity
#N's
Mispriming
Sequence Quality
End Sequence Quality
Position Penalty
End Stability

Objective Function Penalty Weights for Primer Pairs

Product Size Lt: Gt:
Product Tm Lt: Gt:
Tm Difference
Any Complementarity
3' Complementarity
Pair Mispriming
Primer Penalty Weight
Hyb Oligo Penalty Weight

Hyb Oligo (Internal Oligo) Per-Sequence Inputs

Hyb Oligo Excluded Region:

Hyb Oligo (Internal Oligo) General Conditions

Hyb Oligo Size: Min Opt Max
Hyb Oligo Tm: Min Opt Max
Hyb Oligo GC% Min: Opt: Max:
Hyb Oligo Self Complementarity: Hyb Oligo Max 3' Self Complementarity:
Max #Ns: Hyb Oligo Max Poly-X:
Hyb Oligo Mishyb Library: Hyb Oligo Max Mishyb:
Hyb Oligo Min Sequence Quality:
Hyb Oligo Salt Concentration: Hyb Oligo DNA Concentration:

Objective Function Penalty Weights for Hyb Oligos (Internal Oligos)

Hyb Oligo Tm Lt: Gt:
Hyb Oligo Size Lt: Gt:
Hyb Oligo GC% Lt: Gt:
Hyb Oligo Self Complementarity
Hyb Oligo #N's
Hyb Oligo Mishybing
Hyb Oligo Sequence Quality

Acknowledgments

This batch primer design web site was developed by Frank You based on the Primer3 and its web software. The original Primer3 web software is provided by Steve Rozen steve@genome.wi.mit.edu and Whitehead Institute/MIT Center for Genome Research. The development of Primer3 and the original Primer3 web site was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute. under grants R01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander).

The original design of this primer-picking web site by Richard Resnick Primer3's design is heavily based on earlier implementations of similar programs: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander) and Primer v2 (Richard Resnick). Lincoln Stein championed the use of the Boulder-IO format and the idea of making the Primer3 engine a software component.

This page is maintained by frankyou@pw.usda.gov. 
(batch_primer_design.cgi v 0.1 1a)