Primer Design

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.
Overlap Junction List		E.g. 27 requires one primer to overlap the junction between positions 27 and 28. Or mark the source sequence with -: e.g. ...ATCTAC-TGTCAT.. means that primers must overlap the junction between the C and T.
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.
Pair OK Region List		See manual for help.
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
Internal Oligo Excluded Region

Force Left Primer Start		Force Right Primer Start
Force Left Primer End		Force Right Primer End

Sequence Quality

Min Sequence Quality

Min End Sequence Quality

Sequence Quality Range Min

Sequence Quality Range Max

General Primer Picking Conditions

Upload the settings from a file

Primer Size	Min	Opt	Max
Primer Tm	Min	Opt	Max	Max Tm Difference	Table of thermodynamic parameters Breslauer et al. 1986 SantaLucia 1998
Product Tm	Min	Opt	Max
Primer GC%	Min	Opt	Max

Product Size Ranges

Number To Return		Max 3' Stability
Max Library Mispriming		Pair Max Library Mispriming

Thermodynamic Secondary Structure Alignments Use Thermodynamic Oligo Alignment TH: Max Self Complementarity TH: Max 3' Self Complementarity TH: Max Pair Complementarity TH: Max 3' Pair Complementarity TH: Max Primer Hairpin

Old Secondary Structure Alignments Max Self Complementarity Max 3' Self Complementarity Max Pair Complementarity Max 3' Pair Complementarity

Thermodynamic Template Alignments Use Thermodynamic Template Alignment TH: Max Template Mispriming TH: Pair Max Template Mispriming

Old Template Alignments Max Template Mispriming Pair Max Template Mispriming

Max #N's accepted			Max Poly-X
Inside Target Penalty			Outside Target Penalty			Note: you can set Inside Target Penalty to allow primers inside a target.
First Base Index			CG Clamp
Max GC in primer 3' end
3' End Distance Between Left Primers			3' End Distance Between Right Primers
5 Prime Junction Overlap			3 Prime Junction Overlap			(Distance of the primer ends to one overlap position.)
Concentration of Monovalent Cations			Salt Correction Formula		Schildkraut and Lifson 1965 SantaLucia 1998 Owczarzy et. 2004
Concentration of Divalent Cations			Concentration of dNTPs
Annealing Oligo Concentration			(Not the concentration of oligos in the reaction mix but of those annealing to template.)
Sequencing Spacing			Sequencing Interval
Sequencing Lead			Sequencing Accuracy

Liberal Base	Show Debuging Info	Treat ambiguity codes in libraries as consensus
Lowercase masking	Pick anyway	Print Statistics

Objective Function Penalty Weights for Primers

Size	Lt		Gt
Tm	Lt		Gt
GC%	Lt		Gt

TH: Self Complementarity TH: 3' End Self Complementarity TH: Hairpin TH: Template Mispriming

Self Complementarity 3' End Self Complementarity Template Mispriming

#N's
Library Mispriming
Sequence Quality
End Sequence Quality
Position Penalty
End Stability
Primer failure rate

Objective Function Penalty Weights for Primer Pairs

Product Size	Lt		Gt
Product Tm	Lt		Gt

TH: Any Complementarity TH: 3' End Complementarity TH: Template Mispriming Weight

Any Complementarity 3' End Complementarity Template Mispriming Weight

Tm Difference
Library Mispriming
Primer Penalty Weight
Internal Oligo Penalty Weight

Internal Oligo (Hyb Oligo) General Conditions

Internal Oligo Size	Min	Opt	Max
Internal Oligo Tm	Min	Opt	Max
Internal Oligo GC%	Min	Opt	Max

TH: Internal Oligo Self Complementarity TH: Internal Oligo Max 3' End Self Complementarity TH: Internal Oligo Hairpin

Internal Oligo Self Complementarity Internal Oligo Max 3' End Self Complementarity

Internal Oligo Min Sequence Quality
Max #Ns				Internal Oligo Max Poly-X
Internal Oligo Mishyb Library		NONE HUMAN RODENT_AND_SIMPLE RODENT DROSOPHILA		Internal Oligo Max Library Mishyb
Internal Oligo Conc of monovalent cations				Internal Oligo DNA Concentration
Internal Oligo conc of divalent cations				Internal Oligo [dNTP]

Objective Function Penalty Weights for Internal Oligos (Hyb Oligos)

Internal Oligo Size	Lt		Gt
Internal Oligo Tm	Lt		Gt
Internal Oligo GC%	Lt		Gt

TH: Internal Oligo Self Complementarity TH: Internal Oligo 3' End Complementarity TH: Internal Oligo Hairpin

Internal Oligo Self Complementarity Internal Oligo 3' End Complementarity

Internal Oligo #N's
Internal Oligo Library Mishybing
Internal Oligo Sequence Quality
Internal Oligo Sequence End Quality