PCR Primer Analysis

What is a Primer?

A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing. These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal.

PCR Protocols

1. Mix the DNA polymerase, dNTP, DNA templates and DNA primers in a clean PCR tube. Then put the tube in PCR machine and begin setting
2. Pre-Denaturation: 95°C for 10min
3. Denaturation: 95°C for 30s
4. Annealing: 55°C±10°C (The exact temperature is determined by the length and GC percentage of your primers. You could find an answer in the user manual attached with your purchased primers)
5. Elongation: 72°C (The exact temperature is determined by the DNA polymerase you will use. You could find an answer in the user manual attached with the DNA polymerase you have purchased)
6. Cycle: Set the PCR machine to cycle from step 2 to step 4 between 20 and 30 times
7. Final Elongation: 72°C for 10-15min (The exact time is determined by the length of the double strain DNA you want to amplify. Typically, a longer DNA you want to amplify, a longer time you will need to set in this step)
8. Hold: 4°C for a long enough time until you proceed using PCR products in the next experiment

Analysis of Primer Sequences

When designing primers for PCR, sequencing or mutagenesis it is often necessary to make predictions about these primers, for example melting temperature (Tm) and propensity to form dimers with itself or other primers in the reaction. The following program will perform these calculations on any primer sequence or pair.

The programs will calculate both the Tm of the primers, as well as any undesirable pairings of primers. When primers form hairpin loops or dimers, less primer is available for the desired reaction.

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