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Primer3Plus pick primers from a DNA sequence	More...	Source Code
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Load server settings:   Default qPCR Probe p3web v0.4.0 Def p3 v1.1.4 Def   Task:  generic pick_cloning_primers pick_discriminative_primers pick_sequencing_primers pick_primer_list check_primers

Select primer pairs to detect the given template sequence. Optionally targets and included/excluded regions can be specified. Mark an included region to pick primers 5' fixed at its the boundaries. The quality of the primers might be low. Mark an target to pick primers 3' fixed at its the boundaries. The quality of the primers might be low. Pick a series of primers on both strands for sequencing. Optionally the regions of interest can be marked using targets. Returns a list of all possible primers the can be designed on the template sequence. Optionally targets and included/exlcuded regions can be specified. Evaluate a primer of known sequence with the given settings.

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• Main
• General Settings
• Advanced Settings
• Internal Oligo
• Penalty Weights
• Advanced Sequence

Sequence Id:

Paste template sequence below	Or upload sequence file:

Mark selected region:

Excluded Regions:

<

>

Targets:

[

]

Included Region:

{

}

Primer overlap positions:

-

Pair OK Region List:

Pick left primer

Pick hybridization probe

Pick right primer

or use left primer below.	(internal oligo) or use oligo below.	or use right primer below (5'->3' on opposite strand).

© by A. Untergasser et al.

Force Left Primer Start:		Force Right Primer Start:
Force Left Primer End:		Force Right Primer End:
First Base Index:		Sequence Quality:
Start Codon Position:
Min Sequence Quality:
Min End Sequence Quality:
Sequence Quality Range Min:
Sequence Quality Range Max:

© by A. Untergasser et al.

Product Size Ranges

Primer Size	Min:	Opt:	Max:
Primer Tm	Min:	Opt:	Max:	Max Tm Difference:
Primer GC%	Min:	Opt:	Max:

Concentration of monovalent cations:		Annealing Oligo Concentration:
Concentration of divalent cations:		Concentration of dNTPs:

Mispriming/Repeat Library: NONE HUMAN RODENT_AND_SIMPLE RODENT DROSOPHILA

Load and Save

To upload or save a settings file from your local computer, choose here:

© by A. Untergasser et al.

Max Poly-X:		Table of thermodynamic parameters:	Breslauer et al. 1986 SantaLucia 1998
Max #N's:		Salt correction formula:	Schildkraut and Lifson 1965 SantaLucia 1998 Owczarzy et. 2004
CG Clamp:		Use Thermodynamic Primer Alignment:	Activates Settings Starting with TH:
Max End GC:		Use Thermodynamic Template Alignment:	Activates TH: Settings-VERY SLOW
Number To Return:		Max End Stability:
5 Prime Junction Overlap:		3 Prime Junction Overlap:
Min Left Primer End Distance:		Min Right Primer End Distance:
Max Self Complementarity:		Max Pair Complementarity:
TH: Max Self Complementarity:		TH: Max Pair Complementarity:
Max End Self Complementarity:		Max Pair End Complementarity:
TH: Max End Self Compl.:		TH: Max Pair End Complementarity:
TH: Max Hairpin:
Max Template Mispriming:		Pair Max Template Mispriming:
TH: Max Template Mispriming:		TH: Pair Max Template Mispriming:
Max Library Mispriming:		Pair Max Library Mispriming:
Primer Must Match 5 Prime:		Internal Must Match 5 Prime:
Primer Must Match 3 Prime:		Internal Must Match 3 Prime:
Left Primer Acronym:		Internal Oligo Acronym:
Right Primer Acronym:		Primer Name Spacer:

Product Tm	Min:	Opt:	Max:
Product Size	Min:	Opt:	Max:	Debug Information

Pick Anyway     Liberal Base     Do not treat ambiguity codes in libraries as consensus     Use Lowercase Masking

Sequencing

Lead		Spacing
Accuracy		Interval

© by A. Untergasser et al.

Excluded Region:

Hyb Oligo Size:	Min:	Opt:	Max:
Hyb Oligo Tm:	Min:	Opt:	Max:
Hyb Oligo GC%	Min:	Opt:	Max:

Hyb Oligo Monovalent Cations Concentration:		Hyb Oligo DNA Concentration:
Hyb Oligo Divalent Cations Concentration:		Hyb Oligo dNTP Concentration:
Max #Ns:		Hyb Oligo Max Poly-X:
Hyb Oligo Self Complementarity:		Hyb Oligo Max End Self Complementarity:
TH: Hyb Oligo Self Complementarity:		TH: Hyb Oligo Max End Self Complementarity:
TH: Hyb Oligo Max Hairpin:		Max Library Mishyb:
Hyb Oligo Mishyb Library:	NONE HUMAN RODENT_AND_SIMPLE RODENT DROSOPHILA
Hyb Oligo Min Sequence Quality:

© by A. Untergasser et al.

For Primers

For Internal Oligos

For Primer Pairs

Size

Lt:

Gt:

Size

Lt:

Gt:

Product Size

Lt:

Gt:

Tm

Lt:

Gt:

Tm

Lt:

Gt:

Tm Difference

GC%

Lt:

Gt:

GC%

Lt:

Gt:

Product Tm

Lt:

Gt:

Template Mispriming				Template Mispriming
TH: Template Mispriming				TH: Template Mispriming
Library Mispriming		Library Mishyb		Library Mispriming
Self Complementarity		Self Complementarity		Pair Complementarity
TH: Self Complementarity		TH: Self Complementarity		TH: Pair Complementarity
End Self Complementarity		End Self Complementarity		Pair End Complementarity
TH: End Self Complementarity		TH: End Self Complementarity		TH: Pair End Complementarity
TH: Hairpin		TH: Hairpin
#N's		Hyb Oligo #N's
Sequence Quality		Sequence Quality
End Sequence Quality		End Sequence Quality
Position Penalty
End Stability				Primer Penalty Weight
Inside Target Penalty:				Hyb Oligo Penalty Weight
Outside Target Penalty:
Set Inside Target Penalty to allow primers inside a target.

© by A. Untergasser et al.