Primer3Plus - Pick Primers

Primer3Plus pick primers from a DNA sequence	More...	Source Code
Primer3Plus pick primers from a DNA sequence	Help	About

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Main
General Settings
Advanced Settings
Internal Oligo
Penalties
Advanced Seq.
D
Results

Sequence Id:

Paste template sequence below or upload sequence file:

Mark selected region:

			Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list of start,length. 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 template sequence with < and >: e.g. ...ATCT<CCCC>TCAT.. forbids primers in the central CCCC. If one or more Targets is specified then a primer pair must flank at least one of them. The value should be a space-separated list of start,length pairs. E.g. 50,2 requires primers to surround the 2 bases at positions 50 and 51. Or mark the template sequence with [ and ]: e.g. ...ATCT[CCCC]TCAT.. means that primers must flank CCCC. A sub-region of the given sequence in which to pick primers. The value for this parameter has the form start,length. E.g. 20,400: only pick primers in the 400 base region starting at position 20. Or use { and } in the template sequence to mark the beginning and end of the included region: e.g. in ATC{TTC...TCT}AT the included region is TTC...TCT. A list of positions in the given sequence. The value for this parameter has the form position. E.g. 120: only pick primers overlaping the position 120. Or use - in the template sequence to mark the position. Primer3 tries to pick primer pairs were the forward or the reverse primer overlaps one of these positions. This tag allows detailed specification of possible locations of left and right primers in primer pairs. The associated value must be a semicolon-separated list of <left_start>,<left_length>,<right_start>,<right_length> quadruples. Please consult the help for detailed instructions.
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).

5' Overhang:		5' Overhang:

Product Size Ranges

Primer Size	Min:	Opt:	Max:
Primer Tm	Min:	Opt:	Max:	Max Tm Difference:
Primer Bound%	Min:	Opt:	Max:	Annealing Temp:
Primer GC%	Min:	Opt:	Max:

Concentration of monovalent cations:		ANNEALING Oligo Concentration:		Not the concentration of oligos in the reaction mix!
Concentration of divalent cations:		Concentration of dNTPs:
DMSO Concentration:		Formamide Concentration:
DMSO Factor:

Mispriming/Repeat Library:

Load and Save

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

Load only Settings from file:

Load everything from file:

Max Poly-X:		Table of thermodynamic parameters:
Max #N's:		Salt correction formula:
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:		Primer Must Match 3 Prime:
Left Primer Acronym:		Internal Oligo Acronym:
Right Primer Acronym:		Primer Name Spacer:

Product Tm	Min:	Opt:	Max:	Show Secondary Structures
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		primer to readable sequence	Spacing		fw-primer to fw-primer
Accuracy		flexibility of primer position	Interval		fw-primer to rv-primer

Excluded Regions for Internal Oligo:
Internal Oligo Overlap Positions:

Hyb Oligo Size:	Min:	Opt:	Max:
Hyb Oligo Tm:	Min:	Opt:	Max:
Hyb Oligo Bound%:	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:
Hyb Oligo DMSO Concentration:		Hyb Oligo Formamide Concentration:
Hyb Oligo DMSO Factor:
Max #Ns:		Hyb Oligo Max Poly-X:
Rev Comp G rich:		No 5 Prime G:
Hyb Oligo 5 Prime Junction Overlap:		Hyb Oligo 3 Prime Junction Overlap:
Min Hyb Oligo End Distance:
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:
Must Match 5 Prime:		Must Match 3 Prime:
Hyb Oligo Mishyb Library:
Hyb Oligo Min Sequence Quality:

For Primers

For Internal Oligos

For Primer Pairs

Tm

Tm

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.

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

Primer3 Debug Information

Primer3 Input:

Primer3 Output:

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