Am I supposed to first design my primers to get the closest matching Tm and then add in the restriction sites afterwards, or are the restriction sites supposed to be added to my primers first before I calculate the Tm of the whole primers?
In the first cycles, your primer pairs with the original template that doesn't have the restriction sites. But in the later cycles, most of the primer pairs with your PCR product, which does have the sites added. So the formally correct way to do this is to run the first few cycles at the lower tm (without the restriction sites) and the majority of the later cycles with the high tm (with the restriction sites). I've had a few reactions where it actually did make a difference to the outcome and have been routinely using the step-up technique ever since.
I guess you need to only consider the sequence that perfectly matches your target sequence. Please check how far the additional restriction sites do match the target sequence. If not at all, the Tm is only related to the primer sequence. If partially, the original Tm value might be slightly affected.
Exactly as mentioned above; I always use only the nucleotides that basepair with the template to calculate the Tm. This works seamlessly.
In the first cycles, your primer pairs with the original template that doesn't have the restriction sites. But in the later cycles, most of the primer pairs with your PCR product, which does have the sites added. So the formally correct way to do this is to run the first few cycles at the lower tm (without the restriction sites) and the majority of the later cycles with the high tm (with the restriction sites). I've had a few reactions where it actually did make a difference to the outcome and have been routinely using the step-up technique ever since.
1. I only calculated the Tm from the primer sequences specific to the DNA fragment you want to amplified, not including the added 'restriction site'. They worked for me.
2. Besides, documents have suggested that one should also include a few extra bases outside the added 'restriction site' to ensure they can be properly cut by RE before you clone it into the vector. I used 3 extra bases. See attached chart from NEB's suggestion.
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
It is also important to consider that the melting point of the primers will vary depending on the stage of the reaction.
During the first two rounds of the PCR reaction the primers will only bind the target with a sub-section of the primer, however, once the new DNA (with restriction sites on the ends) has been produced after the first few rounds of the reaction, the primers will bind along their whole length (with a higher Tm).
Normally, this is not a problem and a single melting point (Tm without restriction site) can be used, but if the amplification is inefficient, perhaps start with a lower melting point for the first few rounds of the reaction.
Cheers!!
You should consider the sequence of primer that matches to target sequence of DNA sample and calculate the Tm for it. The restriction site dosent match with your target sequence.
You only need to take all the nucleic acids (NA) that complementary to your template into your consideration when you calculate your Tm. All the other extra NA that you added to create digestion sites and protective NA should not be counted in since they should not pair with your template and hence not contribute to the Tm.
Right to do it with only the nucl sequence matching the template for a good Tm calculation. Don't worry about the restriction site which does not match the target anyway. I have done this with and without extra nucl after the restriction site and it works well in both cases.
@yuan I remember my PI opening a NEB restriction enzyme manual with the recommended DNA overhangs for each specific restriction enzyme. Does anyone have an online version of this?
Because the version from the NEB website did not cover everything i.e. recommended length = 5 bp but they only showed maximum 3 bp for EcoRI
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