Hi Agnieszka,

this could also be helpful for calculate the Tm: http://bisearch.enzim.hu/?m=temp

Normally I use also primer 3 to design my primers.

Cheers, Nadine

Hello Agnieszka, I use OligoCalc too, but it has a major drawback, since it lacks the option to compare the properties of a selected primer pair (e.g. for self- and hetero-dimerization). A tool which is able to calculate this is for example OligoAnalyzer (http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/).

For designing qPCR-primers I also use Primer3 (http://bioinfo.ut.ee/primer3/)

Thank you Alexander!

Hi Agnieszka,

this could also be helpful for calculate the Tm: http://bisearch.enzim.hu/?m=temp

Normally I use also primer 3 to design my primers.

Cheers, Nadine

I use Netprimer from last 8 years or so and it really works good as a free online applet. you will have to create a free log in.

http://www.premierbiosoft.com/netprimer/netprlaunch/Help/xnetprlaunch.html

Problem is that every program gives you a different result. The only way is to design a set of assays using 2-3 oligo sets per assay and then use an instrument such as the BioRad CFX to empirically determine the optimal Ta, which is what you are interested in.

Thank you Professor Bustin.

I agree with your commentary, temperatures calculated with available software are theoretical, therefore empirical determination of annealing temperature is essential. However, determination of Tm with oligonucleotides properties calculators, enables me to estimate the temperature gradient for the experiment when setting Ta of primers and in large part, allows to minimize the cost of an experiment.

I am also the user of BioRad CFX thermocyclers: Connect and Touch.

Again, thank you for your notice. I’m glad my question have drawn your attention.

I use primer3 to design primers too, which works fine pretty much most of the time

I use IDT online tools:

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

NCBI web page - Primer-blast...

Hi,

I'be been using Primer 3 (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/). This gives you various primer pairs and their melting temperatures. You can also set parameters to best fit the needs of your target sequence.

Then I would use UCSC's In-silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start) to check my primers. Its quick and easy, but I've been having some trouble with this website. NCBI's primer blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) is always a great and comprehensive way to check your primers before you order anything.

I use Oligo software (Molecular Biology Insights, Inc. Cascade, CO 80809, USA) it is simple and quite useful for primers and real time probes design

Dear,

yup there is always different Tm when calculating by different tools but my experienced is pretty good with JustBio Calculator and Primer3. You may compare Tm from them.be happy

Hello, I use AmpliFX: http://crn2m.univ-mrs.fr/AmplifX?lang=fr

Free, easy to use, and works fine both on PC and Mac

Regards

there's a program called OligoAnalyzer which is free and pretty decent. Using it, you can check secondary structure formation for your oligos, primer pair interactions as well as interactions of different permutations and combinations of all your primers for a multiplex. It also gives you free energy values for each interaction along with other basic info like Tm under different conditions, compliment sequences, GC content etc. This program alongwith online tools like northwestern/IDT/ Primer Blast works pretty well.

I use primer select software, since it give you detailed information and possible secondary structures and such like. The applied biosystem tm calculator works well also

We used http://www.basic.northwestern.edu/biotools/OligoCalc.html and found it as an acceptable program