12/12/2023 0 Comments Snapgene overlapping primers![]() There are a few general rules for primer design: For simple Gibson assembly reactions, overlapping tails from 15 to 30 nucleotides are sufficient. Only when you have no flexibility in your choice of primers, I would use SnapGene to design them. Overlaps need to anneal efficiently at the reaction temperature of 50C. As your fragment length increases, you should increase the length of the overlapping tail. 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. For example, traditional protocols require that all sequence alterations be embedded within the primer itself, which makes it difficult to make insertions >30 nt. ![]() For this, our choice of primers is restricted: the forward primer needs to start with the Rab5 start codon so its location is fixed. Traditional overlap extension PCR mutagenesis protocols remain limited in several critical ways, especially when it comes to generating insertions and deletions. The forward primer should also contain a BspE1 restriction site to fuse the gene to AcGFP1. We want primers with a Tm in the range of 60-62☌. 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. Go back to the Sequence view of mRFP1-Rab5 and select a sequence that starts with the ATG start codon of Rab5. An equivalent procedure, using overhang-based fusion/recircularization (Gibson or NEBuilder methods) can also be simulated in SnapGene. This lesson instructs on how to simulate Inverse PCR and recircularization by blunt end ligation. Select to make a primer for the Top strand: As you elongate your selection, SnapGene automatically displays the length and Tm of the selection: Use Option 1 below to amplify/linearize an entire plasmid, or Option 2 to amplify/linearize a part of a plasmid. 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. The sequence of the site is now added to the 5' end of the primer (blue). 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. 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. This means we can use the existing BamH1 site right downstream of the rab5 gene. So the only requirements of the reverse primer are its location (directly after the stop codon) and its Tm (60-62☌). 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. 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. If you are happy with the sequence of the primer click Add primer to template. ![]() Instead, we recommend that when a polymerase such as Phusion® or Phire® or Q5® is used, the annealing temperature should be about 6–12☌ above the primer T m as calculated by our software.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. The SnapGene team has decided not to provide inaccurate T m values. NEB then recommends using an annealing temperature 0-3☌ above the inaccurately high T m values, or about 6–12☌ above the actual T m. Those older parameters are less accurate, and they tend to give T m values about 6–9☌ higher than the newer parameters. The approach recommended by NEB and Thermo Scientific is to use older thermodynamic parameters to calculate the T m for polymerases such as Phusion®. The reason is that these polymerases are fused to a processivity–enhancing dsDNA-binding domain that stabilizes the primer-template complex. For most PCR polymerases, the optimal annealing temperature for the PCR reaction is about 0–5☌ below the T m for the primers.įor some PCR polymerases such as Phusion®, Phire®, and Q5®, the optimal annealing temperature is about 6‑12☌ above the primer T m.
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