I have around 500 primers set obtained after miSeq run and want to validate best 20 on the basis of primer properties. May someone can suggest what will be the best procedure to rank these 500 to get the top 20 for further validation in Lab
I'd rank based on hair-pin potential, complimentarity and potential for self annealing. Finally use annealing temp to decide the final 20.
You may find this a good tool: http://www.basic.northwestern.edu/biotools/oligocalc.html
regards
If you are going to use them on a very large template (like total genomic DNA) you may also want to check for specificity.
Dear,
There are several parameters you can check with them:
1. check flexibility region ( repetitive nucleotides) like G-C rich region and mask it by RepeatMasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker)
2. You have to check this primers have secondary structure to prevent primer-dimer by http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do
3. calculate Tm, from https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator or manually by this formula: Tm = 4(G + C) + 2(A + T)°C . The best value should be in between 52-58°C.
4. Calculate annealing temperature by this formula : Tm-5
Kindly write to me for any further discussion
Thank you
- Badges
- Science method