In Silico PCR


Help

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.

Designing Primers

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"

Using the Interface

Once you have designed or selected your primers (e.g Primer3), three inputs are required:

Remember to always input primers in 5' to 3' direction and 10 nucleotides is the minimum primer length required. Furthermore, always select the organism name from the given suggestions to avoid errors.
All three fields are required to proceed with amplification.

Advanced Options

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

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

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.

Results

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:
1Name of sequence in NCBI2GI number
3Accession number4nucleotide/base size of the PCR product
5location from where amplification begins6location from where amplification end
7FP: Forward Primer8RP: Reverse Primer
9Views amplicon sequence in fasta format10Download amplicon in fasta format
11Shows your amplicon in NCBI Sviewer


This web-server was designed by Gaurav Panchal and is provided by the Department of Biological and Environmental Sciences at the University of Hertfordshire, Hatfield, UK.
For any queries email g.panchal@herts.ac.uk.