The thermodynamics of DNA structural motifs. Our algorithm uses accurate parameters for deoxyinosine (I), and assumes that deoxyuracil (U) will give the same results as thymidine (T), but other non-DNA bases are not supported.Salt-corrected Tm estimates for a specific buffer can be obtained using the IDT Oligo Analyzer. The monovalent cation concentration in PCR mixtures can vary, and Mg2+ also affects the Tm.More sophisticated multi-state annealing simulations are offered by the UNAFold Web Server or the commercial Visual OMP software. Structures such as hairpin loops are not considered. Our algorithm employs a two-state melting model, which typically works best for short oligos.If a duplex contains loops, the Tm value is only an approximation.For oligos with degenerate (mixed) bases, the Tm is estimated by averaging the relevant parameters.SnapGene assumes an oligo concentration of 0.25 μM for a PCR primer, or 0.5 μM each for two annealed oligos. The Tm also depends on the oligo concentration.This convention is used by many oligo suppliers. SnapGene assumes a Na+ concentration of 50 mM. The Tm depends on the salt concentration.Because detailed duplex structures can be distracting, SnapGene shows simplified duplexes by default, but you can see full duplex structures with a setting, see Show Full Structures for Primer-Template Duplexes. For a given duplex, the calculation accounts for any internal mismatches, loops, and dangling ends.įor a primer binding site, nearest neighbor calculations are combined with dynamic programming to find the most energetically favorable duplex. This method is the most accurate one available. If you want to design primers for other applications where efficiency and specificity of the primers can be taken into account, you shouldn't use SnapGene, use CLC Main Workbench or Primer-Blast instead.SnapGene calculates oligo melting temperature (Tm) values using a nearest neighbor thermodynamic algorithm with up-to-date parameters. If you are happy with the sequence of the primer click Add primer to template. Select to make a primer for the Bottom strand: To transform the selection into a primer expand Primers in the top menu and select Add primer. Select a sequence that starts after the stop codon of Rab5 with the correct Tm: Try to do the exercise without peeking at the solution. So the only requirements of the reverse primer are its location (directly after the stop codon) and its Tm (60-62☌). This means we can use the existing BamH1 site right downstream of the rab5 gene. If you are happy with the sequence of the primer click Add primer to template (green).įor cloning Rab5 into the vector later on, we will use a BamHI restriction site. To do this click at the 5’ end of the primer and type a random sequence of 6 nt (red): To shield the site we will some random nucleotides to the 5' end of the primer. The sequence of the site is now added to the 5' end of the primer (blue). Select BspE1' in the site box (red) and click the Insert button (green): To add the restriction site select the Insertions tab: You can just type the sequence you want to add.You can insert codons, restriction sites and tags.There are two ways to add a sequence to the primer: To clone the gene you need a BspE1 site at the 5' end of the primer. Select to make a primer for the Top strand: As you elongate your selection, SnapGene automatically displays the length and Tm of the selection: Go back to the Sequence view of mRFP1-Rab5 and select a sequence that starts with the ATG start codon of Rab5. The only thing that is still variable in this case is the length of the primer, which will be determined by the Tm that we wish. We want primers with a Tm in the range of 60-62☌. The forward primer should also contain a BspE1 restriction site to fuse the gene to AcGFP1. For this, our choice of primers is restricted: the forward primer needs to start with the Rab5 start codon so its location is fixed. For cloning this is often the case.įor instance, we want to make primers to clone the Rab5 gene from plasmid mRFP1-Rab5 as a fusion to the AcGFP1 gene in plasmid pAcGFP1-C1. Only when you have no flexibility in your choice of primers, I would use SnapGene to design them.
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