Primer Tm estimation methods: Difference between revisions
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(ficticous primer) |
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! style="background:lightgrey"|Breslauer '86 | ! style="background:lightgrey"|Breslauer '86 | ||
! style="background:lightgrey"|SantaLucia '98 | ! style="background:lightgrey"|SantaLucia '98 | ||
|- | |||
| 50/50 mixed: AGAGAGAGAGAGAGAGAGAG | |||
| 60 | |||
| 52 | |||
| 46.3 | |||
| 47.7 | |||
|- | |||
| 50/50 separated: AAAAAAAAAAGGGGGGGGGG | |||
| 60 | |||
| 52 | |||
| 66.0 | |||
| 52.7 | |||
|- | |- | ||
| ActB F: TTGCTGACAGGATGCAGAAG | | ActB F: TTGCTGACAGGATGCAGAAG | ||
Line 41: | Line 53: | ||
: [http://www.basic.northwestern.edu/biotools/oligocalc.html online tool] using Wallace formula for oligos >13 | : [http://www.basic.northwestern.edu/biotools/oligocalc.html online tool] using Wallace formula for oligos >13 | ||
* Breslauer et al. 1986, DOI:10.1073/ pnas.83.11.3746 | * Breslauer et al. 1986, DOI:10.1073/ pnas.83.11.3746 combined with Schildkraut et al. 1965 salt correction formulae | ||
: [http://frodo.wi.mit.edu/primer3/ Primer3] and [http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Primer3Plus] default maintained for backwards compatibility | : [http://frodo.wi.mit.edu/primer3/ Primer3] and [http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Primer3Plus] default maintained for backwards compatibility | ||
* SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 | * SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 thermodynamics & salt correction | ||
: Primer3 recommended setting; also default settings of the NCBI's [http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer BLAST] | : Primer3 recommended setting; also default settings of the NCBI's [http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer BLAST] | ||
Revision as of 01:45, 28 September 2009
example primer | Marmur rule | Wallace rule | Breslauer '86 | SantaLucia '98 |
---|---|---|---|---|
50/50 mixed: AGAGAGAGAGAGAGAGAGAG | 60 | 52 | 46.3 | 47.7 |
50/50 separated: AAAAAAAAAAGGGGGGGGGG | 60 | 52 | 66.0 | 52.7 |
ActB F: TTGCTGACAGGATGCAGAAG | 60 | 52 | 60.1 | 52.4 |
ActB R: TGATCCACATCTGCTGGAAG | 60 | 52 | 59.8 | 51.5 |
Tubb5 F: GATCGGTGCTAAGTTCTGGGA | 64 | 54 | 61.5 | 53.7 |
Tubb5 R: AGGGACATACTTGCCACCTGT | 64 | 54 | 60.8 | 55.1 |
- Marmur formula: Tm = 4 x GC + 2 x AT
- not recommended for more than 13nt; assumes 50mM monovalent cations
- Marmur J and Doty P (1962) J Mol Biol 5:109-118; PMID 14470099
- Wallace formula: Tm = 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)
- Wallace RB et al. (1979) Nucleic Acids Res 6:3543-3557, PMID 158748
- online tool using Wallace formula for oligos >13
- Breslauer et al. 1986, DOI:10.1073/ pnas.83.11.3746 combined with Schildkraut et al. 1965 salt correction formulae
- Primer3 and Primer3Plus default maintained for backwards compatibility
- SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 thermodynamics & salt correction
- Primer3 recommended setting; also default settings of the NCBI's Primer BLAST