In Silico PCR enables you to calculate PCR results theoretically using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome. This page guides you about how to use the interface and understand the results.
Current interface does not design primers for you. If you wish to design primers you can browse to primer3.ut.ee insert the DNA sequence you wish to obtain primers and "pick primers"
Once you have designed or selected your primers (e.g Primer3), three inputs are required:
The advanced option parameters are related to experimental conditions which can further optimise the PCR. These conditions are related to the reaction volume and number of cycles to be performed.
KCL is a salt contained in the PCR buffer solution. It facilitates annealing of the primer to the DNA template by reducing the repulsion between the negatively charged phosphate backbone of DNA. KCL therefore stabilises the primer-template binding, and enables the polymerase to start adding nucleotides. 50mM KCL is considered optimal, although some reactions may be more efficient at higher concentrations.
MgCl2 is required as a cofactor for the polymerase to function effectively. Higher Mg concentrations will result in a quicker reaction, although too much can lead to the polymerase making errors. If too little Mg is present, the polymerase will not be activated and the reaction will not proceed as quickly as it should, resulting in low copy numbers.
The tool collects sequences from a nucleotide database and the result of each amplicon is separated by a horizontal line. Below is an example of a single possible amplicon result:
|1||Name of sequence in NCBI||2||GI number|
|3||Accession number||4||nucleotide/base size of the PCR product|
|5||location from where amplification begins||6||location from where amplification end|
|7||FP: Forward Primer||8||RP: Reverse Primer|
|9||Views amplicon sequence in fasta format||10||Download amplicon in fasta format|
|11||Shows your amplicon in NCBI Sviewer|