Why annealing temperature tm

Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Filter using one or more categories to focus on specific topics, or use the search bar to perform a text search. Order by stock part number ». Get Help EN. Submit question. Apply Close. However, the nucleotide sequence analysis of the eight obtained cloned PCR products revealed that six clones harboured false, non- papA inserts.

In both cases even though both primers, forward primer 22 and reverse primer 23, were added to the PCR mixture, the primer 22 was used as the forward but also the reverse primer. The anticipated annealing sites for non-specific papA -primer binding in this case are presented in Figure 3. Anticipated annealing sites for non-specific papA -primer binding in the methylisocitrate lyase gene.

The shown sequences are enumerated according to the CP The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from to nt is shown, and for the reverse primer annealing site, the sequence from to nt is shown. The primer sequence is in the grey box. The methylisocitrate lyase gene is positioned in the deposited sequence from to nt. The anticipated annealing sites for non-specific papA -primer binding in this case are presented in Figure 4.

The shown sequences are enumerated according the CP Seven PCR products, all of the expected size, of around bp, were cloned, and the obtained insert sequences were analysed. The anticipated annealing sites of POP primer on the analysed X The shown sequences are enumerated according the X For the forward primer annealing site, the sequence from 27 to 48 nt is shown, and for the reverse primer annealing site, the sequence from to nt is shown.

The prsE gene is positioned in the deposited sequence from 79 to nt, and the prsF gene is positioned from to nt. Three PCR products, all of the expected size, of around bp, were obtained and cloned, and the obtained insert sequences were analysed.

All three clones harboured the expected papG sequence. The papG gene is positioned in the deposited sequence from to nt. Nucleotide sequence analysis of all eight clones showed that four harboured correct and four harboured false inserts. The anticipated annealing sites for non-specific F17G -primer binding in analysed nucleotide sequences are presented in Figure 7.

Anticipated annealing sites for non-specific F17G -primer binding in the rtn gene. For the forward primer, F17G-1, annealing site, the sequence from to nt is shown, and for the reverse primer, F17G-2, annealing site, the sequence from to nt is shown.

The rtn gene is positioned in the deposited sequence from to nt. The main aim of our research was to determine the sequences of chosen P- and Ffimbriae genes among E.

As we assumed that the fimbriae of such E. Nucleotide sequence analysis revealed that also in the case of papEF clones, even though the cloned inserts were as hoped for fimbrial inserts, even if they were Prs-fimbrial genes, the binding site of the reverse primer was not the expected one. All 16 were cloned and analysed, and all clones were with correct inserts data not shown. To conclude, we all know that with PCR, we can obtain false unspecific products, and we believe that such PCR products will be distinguished from right PCR products, because the false PCR products will not be of the correct expected size; however, our results showed that also PCR products of the expected size can be false PCR products.

The author is very thankful to Wim Gaastra for the primer nucleotide sequences. This analysis was supported by the Slovenian Research Agency P Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers.

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Downloaded: Keywords primer binding annealing temperature sequence analysis Escherichia coli adhesin P-fimbriae Ffimbriae. Introduction Since polymerase chain reaction PCR was invented in the mids, it has made its way into all molecular biology, genetic, microbiology or biochemistry laboratories, where it is, due to its simplicity and efficiency, used in a very wide range of PCR -based techniques and applications [ 1 , 2 ].

Table 1. Table 2. Primers and their melting temperatures Tm used to amplify the studied genes. More Print chapter. How to cite and reference Link to this chapter Copy to clipboard.

Available from:. What I can tell you, is that the Tm really depend on the polymerase you are using for the PCR reaction and for each polymerase there is a set of PCR conditions you have to follow. First of all, it is suggested to use the Finzymes Tm calculator to determine the Tms of your primers. Usually the Tms are higher than the ones calculated by IDT. Then, according to the length of your primers, different annealing temperature should be set:. When calculating the Tm of your primers with the calculator: Fwd primer Tm is You are right that the annealing temperature shouldn't be higher than the Tm of the primers.

I think the problem is that different polymerases and different Tm calculators are used in the papers you are referring to. I can discuss other PCR optimizations but let me know first whether the problem is not just the anneaning temperature. Sign up to join this community. The best answers are voted up and rise to the top. Stack Overflow for Teams — Collaborate and share knowledge with a private group.

Create a free Team What is Teams? Learn more. Does an annealing temp higher than primer's Tm contribute to primer dimer? Ask Question. Asked 9 years, 9 months ago. Active 9 years, 9 months ago. Viewed 18k times. Calculating the Tm via primer blast here are the numbers: Forward Tm: Improve this question. Add a comment. Active Oldest Votes. Improve this answer. Post-doc: scoffs Who do you think we are? This may be a last resort thing and may take a million years but you can always send an e-mail to the authors of those papers and see if they can help you optimize your reactions or share with you how exactly they did it.