| Primer3 (v. 0.4.0) Pick primers from a DNA sequence. | Checks for mispriming in template. | disclaimer | Primer3 Home |
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Paste source sequence 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): NONE HUMAN RODENT_AND_SIMPLE RODENT DROSOPHILA
| Pick left primer, or use left primer below: | Pick hybridization probe (internal oligo), or use oligo below: | Pick right primer, or use right primer below (5' to 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. |
| Number To Return | Max 3' Stability |
| Max Repeat Mispriming | Pair Max Repeat Mispriming |
| Max Template Mispriming | Pair Max Template Mispriming |
General Primer Picking Conditions
| 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: |
| Max Self Complementarity: | Max 3' Self Complementarity: |
| Inside Target Penalty: | Outside Target Penalty: | Note: you can set Inside Target Penalty to allow primers inside a target. |
| First Base Index: | CG Clamp: |
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